[The Enzymes] Volume 7 || 20 β-Galactosidase

P-Galactosidase KURT WALLENFELS RUDOLF WEIL

I . Introduction . . . . . . . . . . . . . 618 11 . Occurrence . . . . . . . . . . . . . 619

I11 . Assay and Standardization . . . . . . . . . . 620 A . Standard Assays and Units of Activity . . . . . 623 B . Transferase Assay . . . . . . . . . . 624 C . Assay of Bacterial Suspensions and Animal Material . . 624

IV . Purification . . . . . . . . . . . . . 624 A . p-Galactosidase from E . coli . . . . . . . 624 B . p-Galactosidases from Other Microorganisms . . . 625 C . Animal p-Galactosidases . . . . . . . . 626

627 A . Size, Shape, and Quaternary Structure . . . . . 627

C . Denaturation and Renaturation Studies . . . . . 634 636

VIII . Enzymic Properties . . . . . . . . . . . 641 A . Specificity . . . . . . . . . . . . 641 B . pH Dependence . . . . . . . . . . 644 C . Metal Activation . . . . . . . . . . 645 D . Kinetics . . . . . . . . . . . . 648 E . Mechanism of Reaction . . . . . . . . 651 F . The Active Site . . . . . . . . . . 657 G . The Transfer Reaction . . . . . . . . . 658 H . Condensation Reactions . . . . . . . . 660 I . Inhibition Studies . . . . . . . . . . 660 J . Other 8-Galactosidases of Microbial, Plant, and

Animal Origin . . . . . . . . . . . 661

V . Physicochemical Properties . . . . . . . . . .

B . Associated Forms of p-Galactosidase . . . . . 633

VII . Immunological Properties . . . . . . . . . . 639 VI . Chemical Properties . . . . . . . . . . . .

617

618 K. WALLENFELS AND R. WEIL

1. Introduction

Oligo- or polysaccharides containing D-galactose joined through a P-glycosidic bond occur in most if not all organisms. Thus, i t is not surprising that the corresponding glycosidases are as widely distributed as their substrates. This universal occurrence coupled with the simple enzymic assay and the availability of numerous substrates have made P-galactosidase one of the most widely studied glycosidases.

It is the purpose of this chapter to present information on the chemistry and enzymology of P-galactosidases (EC 3.2.1.23) from various sources and to cover the development in this field since the second edition of this treatise. The reader will often be referred to the material presented in the previous edition and in a related review on the same topic (1, a ) .

A bewildering amount of new data has been accumulated in the last 10 years, especially in the field of animal P-galactosidase which is still in the stage of rapid and somehow unordered expansion. In the case of I

P-galactosidase from Escherichia coli, that piBce de re’sistance for en- zymologists, emphasis has shifted from analysis of function, such as kinetics and specificity, to studies of structure. One of the great achieve- ments of molecular genetics of the last decade, the general concept of operon structure, expression, and regulation, is based largely on work on the lac operon of E. coli. Data from structural work were essential to confirm and clarify the picture provided by genetical studies.

Thus, isolation and characterization of mutant proteins and their correlation to the structural gene and analysis of in vitro complementation by reassociation of peptide fragments were useful in understanding the structure of the z gene. Much of this work has been reviewed recently by Zabin and Fowler (3) and by Ullmann and Perrin ( 4 ) .

Even though studies have been extensive, many aspects of galactosidase catalysis remain unclear. Little is known about the mechanism of action. Moreover, nothing is known about the active site.

1. K. Wallcnfelw and 0. P. Malhotm. “The Enzymes,” 2nd ed.. Vol. 4, p. 409,

2. K. Wallenfels and 0. P. Malhotra, Advan. Carbohydrate Chem. 16, 239 (1961). 3. I. Zabin and A. V. Fowler, in “The Lactose Operon” (J. R. Beckmith and

D. Zipser, eds.), p. 27. Cold Spring Harbor Monographs, 1970. 4. A. Ullmann and D. Perrin, in “The Lactose Operon” (J. R. Beckwitli and D.

Zipser, eds.), p. 143. Cold Spring Harbor Monographs, 1970.

1960.

20. P-GALACTOSIDASE 619

II. Occurrence

P-Galactosidases have been found in nurr: erous microorganisms, animals, and plants. Since tests for fermentation of lactose play an important role in diagnostic bacteriology of 33nterobacteriaceae) the oc- curreiice of P-galactosidase in gram-negative rods has been extensively investigated (5-10). Enzymic activity was found in several hundred strains of Enterobacteriaceae, in strains of Pseudomonodaceae, Parvo- bacteriaceae, and Neisseriaceae (1 1). Among gram-positive bacteria Diplococcus pneumoniae (12) ) Streptococcus lactis (13) ) Bacillus mega- terium (14) ) Baccilus subtilis (15) ) and seireral Propionibacteria (16) were found to produce p-galactosidase.

Staphylococcus aweus (17 ) and one strain of Streptococcus lactis (18) contain a special phosphogalactosidnee which splits 6-O-phosphoryl P-D- galactosides. These derivatives are synthesized during the passage of P-D-galactosides across the cell membrane by a phosphotransferase system. ,8-Galactosidase activity is present in numerous species of yeast (1, 19-21), protozoa (22, 2k"a), and in various fungi (1, 2 3 ) .

The widespread occurrence of P-galactosidaee in mammalian organs is probably related to the multiple physiological functions of the enzyme.

5. L. Le Minor and F. Ben Hamida, Ann. Znst. l'asteur 102, 276 (1962). 6. S. Saturm-Rubinsten and D. PiCchaud. Ann. Inst. Pasteur 104, 284 (1963). 7. P. Biilow, Acta Pathol. Microbiol. Scctnd. 60, 387 (1964). 8. A. H. Lubin and W. H. Ewing, Public Health Lab. 22, 83 (1964). 9 . R. E . Goodman and M. J. Picket, J . Bacteriol. 92, 318 (1966). 10. J. H. Marymont, U. Amanna, and B. H. Lloyd, Amer. J. C h i . Pathol. 46,

11. W. P. Corbett and B. W. Catlin, J . Hacteriol 95, 52 (1968). 12. R. C. Hughrs and R. W. Jeanloz, Biochemistry 3, 1535 (1964). 13. J. E. Citti, W. E. Sandinc, and P. R. Ellikcr, J . Bacteriol. 89, 037 (1965). 14. 0. E. Landman, B B A 23, 558 (1957). 15. P. J. Anema, BBA 89, 495 (1964). 16. W. Wisniewski, Acta Biochim. Polon. 12, 115 (1965). 17. W. Hengstenberg, J. B. Egan, and M. 1,. Morse, Proc. Nat. Acad. Sci. U. S.

18. L. L. McKay, L. A. Walter, W. E. Sandine, and P. R. Elliker, J . Bacteriol.

19. P. A. J. Gorin, J. F. T. Spencer, and H. J . Phaff, Can. J . Chem. 42, 1341

20. R. Davies, J . Gen. Microbiol. 37, 81 (1964). 21. L. Biermann and M. D. Glante, BRA 167, 373 (1968). 22. G. J. Harrap and W. M. Watkins, BJ 117, 667 (1970). %a. B. H. Howard, BJ 89, 9OP (1963). 23. R. J. W. Byrde and A. H. Fielding, Nature 285, 390 (1965).

702 (1966).

58, 274 (1967).

99, 603 (1969).

(1964).


The role of intestinal P-galactosidasc for the hydrolysis and consequently for the absorption of dietary lactose is well known, and there is now strong evidence that lysosomal P-galactosidasc is a key enzyme in the degradation of glycolipids (24, 25) , mucopolysaccharides, and glycoproteins (25 ) . The occurrence of intestinal P-galactosidase in various animals and the pattern of development during growth have been reviewed by Semenza (26 ) . Information regarding the distribution of lysosomal p-galactosidase is widespread throughout the literature. A com- prehensive list of references has recently been presented by Nisizawa and Hashimoto (27). The digestive juices of several species of snails contain high activities of p-galactosidase (28-30), which is probably necessary for the breakdown of nutritional p-D-galactosides.

In the plant kingdom, high enzymic activities are found in seeds (1, 31) and leaves (32) where the enzyme is probably related to the catabolism of galactolipids, such as p-D-galactosyl diacylglycerol, which are universally distributed in plants and algae.

111. Assay and Standardization

The hydrolysis of j3-D-galactosides by P-galactosidase may be followed by measurement of the liberation of either the glycon or the aglycon. Liberation of galactose, however, is permissible as basis of hydrolase assay only under conditions where no transfer to suitable acceptors occurs. On the other hand, simultaneous determination of liberated galactose and aglycon in the presence of an acceptor allows an exact measurement of transferase activity. Liberation of galactose can be most accurately measured using the enzyme galactose dehydrogenase from Pseudomonas saccharophila (33, 34) or from the related organism

24. J. S. O’Brien, S. Okada, M. W. Ho, D. 1,. Fillerup, M. 1,. Venth, and K. Adams, Federation Pioc. 30, 956 (1971).

25. R. C. Spiro, Ann. Rev. Biochem. 39, 599 (1970). 26. C. Semenza, in “Handbook of Physiology” (Amer. Physiol. SOC., J. Field,

cd.), Sect. 6, Vol. 5, p. 2543. Williams & Wilkins, Baltimore, Maryland, 1968. 27. K. Nisizawa and Y. Hashimoto, in “The Carbohydrates” (W. Pigman, D.

Horton, and A . Herp, eds.), 2nd ed., Vol. 2A, p. 241. Academic Press, New York, 1970.

28. H. J. Horstmann, 2. Physiol. Chem. 337, 57 (1964). 29. J. E. G. Barnett, BJ 96, 72P (1965). 30. R. Got, A. Marnay, P. Jarrige, and J. Font, Nature 204, 686 (1964). 31. K. M. L. Agrnwal and 0. P. Bahl, JBC 243, 103 (1968). 32. S. Gatt and E. A. Baker, BBA 206, 125 (1970). 33. K. Wallenfels and G. Kurz, Biochem. Z. 335, 559 (1962). 34. K. Wallenfels and G. Kurz, “Methods in Enzymology,” Vol. 9, p. 112, 1966.


Pseudomonas fluorescens (34a) . Since fucosc: is an excellent substrate of this enzyme, determination of fucosidase :tctivity is possible as well.

The enzyme galactose oxidase from Dacty!iunz dendroides, which has also been used for determination of free galactose, is rather unspecific and attacks galactosides as well as other derivati1,es of galactose (35) . How- ever, the broad specificity of the enzyme was successfully employed for preparation of tritium-labeled substrate of cerebroside galactosidase from rat brain (36, 37). This method consists of oxidation of the 6 position of the galactose moiety and reduction wi;h tritiated NaBH,; i t may be a useful tool for preparing labeled galactcsides in general.

By using 14C-labeled galactose and a suitable acceptor, several labeled galactosides were synthesized by dircbct combination of the free galactose with the acceptor in the presence of p-galactosidase (38 ) . Other labeled substrates for assayicg special galactosidases have been prepared by chemical synthesis using I4C- acetobromogalactose (39), by tritiation according to Wilzbach (.GO), or by specific enzymic transfer from UDP-14C-gal as a donor ( 4 1 ) .

Most procedures for assaying P-galactosidase are based on the determination of the liberated aglycon. Thus, glucose liberated from lactose can be estimated enzymically using glucose oxidase (42, 43) or hexokinase coupled with glucose 6-phosphate dehydrogcnase (43). In general, for many substrates of p-galactosidase, enzymes, are available that permit a convenient estimation of the aglycon.

The most common substrates for assaying p-galactosidase, however, are chromogenic galactosides. The first of these substrates was p-nitrophenyl p-D-galactoside, introduced by Aizawa (44) . However, only after Lederberg described the use of o-nitrophenyl p-D-galactoside as a sensi-

34a. The enzi me from Pseudomonas fluorexens is now commercially available. 35. G. Avigad, D. Amaral, C. Asensio, and B. C. Horecker, JBC 237, 2736 (1962). 36. A. K. Hajra, D. M. Bowen, Y. Kishimoto, and N. S. Radin, J. Lipid Res. 7,

37. D. M. Bowen and N. S. Radin, BBA 152, 587 (1968). 38. W. Boos, J. Lehmann, and K. 'Wallcnfels, Curbohydrate Res. 7, 381 (1968). 39. R. 0. Brady, A. E. Gal, J. N. Kanfer. and R. M. Bradley, JBC 240, 3766

40. R. 0. Brady, A. E. Gal, R. M. Bradley, and E MBrtensson, JBC 242, 1021

41. S. Okada and J. S. O'Brien, Science 160, 1002 (1968). 42. K. Wallenfels and H. Diekmann, in "Hoppe-Seyler/Thierfelder Handbuch der

physiologisch and pathologisch-chemischen Analyse" (K. Lang and F. Lehnartz, eds.), 10th ed., Vol. 6, Part B, p. 1156, Springer Verlag, Berlin and New York, 1966.

379 (1966).

(1965).

(1967).

43. A. Dahlqvist, Anal. Biochem. 22, 99 (1968). 44. K. Aiaawa, Enzymologia 6, 321 (1939).


tive assay was this simple and convenient assay generally accepted (45). The introduction of this method was certainly an important factor in determining the choice of P-galactosidase from E . coli for studying various aspects of genetics and protein biosynthesis. The sensitivity of the assay was further enhanced by employing radioactive o-nitrophenyl P-D-galactoside labeled with 14C in the aglycon (46). An auto- mated enzyme analysis system has been described and applied for assaying lysosomal galactosidase using p-nitrophenyl p-D-galactoside as a substrate (47).

Cohen e t al. introduced 6-bromo-2-naphthyl P-D-galactoside, which gives rise to insoluble 6-bromo-2-naphthol (48) after enzymic hydrolysis. The amount of liberated aglycon is measured after coupling with a diazonium salt (diazo fast blue). The low solubility of 6-bromo-2- naphthol, on the other hand, makes the galactoside a iraluable tool for histochemical use (49). In addition, it proved to be useful for detecting enzymically active zones after electrophoresis of enzyme preparations on polyacrylamide gels (50, 51). Pearson e t al. studied several substituted indolyl P-D-galactosides for histochemical localization of P-galactosidase and found the 5-bromo-4-chloroindolyl P-D-galactoside especially suitable for this purpose (52). This compound was also used for activity staining after electrophoresis (53) .

Sensitivity of the galactosidase assay was increased considerably by using fluorogenic substrates. Thus, methylumbelliferyl p-D-galactoside was introduced for estimating low enzymic activities (54) and was found useful for assaying samples from animal tissues and urine (55, 56). The method was further adapted as a micromethod using as little as 1 pg of freeze-dried material (57). Another fluorogenic substrate recently

45. J. Lederberg, J . Bacteriol. 60, 381 (1950). 46. H. No11 and J. Orlando, Anal. Biochem. 2, 205 (1961). 47. D. W. Bradley and A. L. Tappel, Anal. Biochem. 33, 400 (1070). 48. R. B. Cohen. K. C. Tsoii. S. H. Rutenburg, and A. M. Srligman, JBC 195,

49. A. M. Rutenburg, S. H. Rutenburg, R. Monis, R. Teagur, and A. M. Selig-

50. S. H. Appel, D. H. Alpers, and G. M. Tomkins, J M B 11, 12 (1965). 51. S. L. Marchesi, E. Steers, and S. Shifrin, BBA 181, 20 (1969) 52. B. Pearson, P. C. Wolf, and J. Vazquez, Lab. Invest. 12, 1249 (19633. 53. R. Got, A. Marnay, and P . Jarrige, Experientin 21, 653 (1965). 54. R . E. MrCaman and E . Robins, J . Newochem. 5, 32 (1959). 55. D. Robinson, R . G . Price, and N. Dance, BJ 102, 525 (1967). 56. D. Robinson, R. G. Price, and N. Dance, BJ 102, 533 (1967). 57. E . Robins, H. E. Hirsch, and S. S. Emmons, JBC 243, 4246 (1968).

239 (1958).

man, J . Hktochem. Cytochem. 6, 122 (1958).


introduced is 2-naphthyl p-D-galactoside (58 ) . Rotman synthesized the fluorogenic substrate fluorescein di-P-D-galactoside (69) and developed an interesting method that permits the assay of single enzyme molecules in droplets of microscopic size (60).

A. STANDARD ASSAYS AND UNITS OF ACTIVITY

Since enzymes from different sources have widely different pH optima and ionic requirements, no general procedure for assaying enzymic activities can be given. Regarding animal galactosidases the reader is referred to the methods described by Dahlqvist (61) and Vaes (62 ) .

P-Galactosidase from 8. coli is usually asayed in phosphate or tris buffers containing mercaptoethanol and optin- a1 concentrations of sodium and divalent ions. However, i t should be kept in mind that all compounds containing hydroxyl groups are glycosyl acceptors that modify the kinetics of the enzyme. It is therefore suggested that the assay be performed in imidazole-HC1 or histidine buffer, which allow the study of ionic requirements as well.

Standard assays of P-galactosidase from E. coli were developed by different groups working on the characterization of the enzyme (63-67). Since some of the test conditions are strikingly different, it is not surprising that definitions of enzymic activity differ considerably. The unit of activity most widely used is that defined by Craven e t al. (68) as the amount of enzyme that hydroiyzes moles of o-nitrophenyl p-D- galactoside in 1 min a t 28°C in 0.1M sodium phosphate buffer (pH 7.0), containing M Pvlg’+, 0.1 M mercriptoethanol and 2.3 X M substrate. However, i t must be emphasized that this unit is not limited to the hydrolytic activity of the enzyme but includes the transferase activity as well.

58. N. G. Asp, BJ 121, 299 (1971). 59. B. Rotman. Proc. Nat. Acad. Sci. U . S. 47, 1981 (1961). 60. B. Rotman, J. A . Zderic, and M. Edelstein, Proc. Na t . Acad. Sci. U . S. 50,

61. A. Dahlqvist, “Methods in Enzymology,” Vol. 8, p. 584, 1966. 62. G. Vnes, “Methods in Enzymology.” Vol. 8, p. 509, 1966. 63. M. Cohn and J. Monod, BBA 7, 153 (1951). 64. S. A. Kuby and H. A. Lardy. JAGS 75, 890 (1953). 65. A. S. L. Hu, R. G. Wolfc, and F. J. Reithel, ABB 81, 500 (1959). 66. K. Wallenfels. M. 1,. Zarnitz, G. Laiile. H. Bender, and M. Kescr, Biochem.

67. K. Wallenfels, “Methods in Enzymology,” Vol. 5, p. 212, 1962. 68. G. R. Craven, E. Steers. and C. B. Anfinsen, JBC 240, 2468 (1965).

1 (1963).

2. 331, 459 (1959).


B. TRANSFERASE ASSAY

Transferase activity of P-galactosidase from E . coli is assayed by comparing the amount of liberated galactose in the presence and absence of glycerol or another suitable galactosyl acceptor (69).

C. ASSAY OF BACTERIAL SUSPENSIONS AND ANIMAL MATERIAL

Determination of activity in bacterial suspensions is usually carried out on toluenized samples, Novick and Weiner (70) obtained the best reproducibility of results by adding sodium deoxycholate and toluene to E . coli suspensions. With lactose-positive Neisseria, 1-butanol was found to be superior to toluene for removing the permeability barrier (11). However, the observed increased activity may in part, result from the transfer reaction in the presence of the alcohol.

Highest activity of P-galactosidase in animal tissues is usually found after homogenizing the material in distilled water or in the presence of detergents such as Triton X 100 or digitonin, or by repeated freezing and thawing (62) . Methods for the solubilization of intestinal p-galactosidase by digestion with proteolytic enzymes have also been described (61).

IV. Purification

A. P-GALACTOSIDASE FROM E. coli

Isolation and purification of P-galactosidase from E . coli is relatively easy. The enzyme represents up to 5% of the total proteins in some constitutive strains of E. coli. In addition, the large size and stability of the enzyme facilitate the procedure of isolation.

The purification of the enzyme was first studied in a systemic way by Cohn and Monod (6.3). Later Wallenfels et al. (66) modified and extended a procedure described by Kuby and Lardy (64) and achieved crystallization of the enzyme from strains M L 35 and 309. Hu et al. crystallized the enzyme from strain M L 308 by using DEAE-cellulose

69. G. Kurz, Ph.D. Thesis, Freiburg (1967). 70. A. Novick and M. Weiner, Proc. N a t . Acnd. Sci. U . S . 43, 553 (1957).


chromatography and preparative electrophoresis (65). Perrin applied a similar procedure but omitted the electroplioresis and reported crystallization of the enzyme from strain K 12, ~ n d of an immunologically related less active protein from a mutant strain (71) .

Recently, Colby and Hu purified and crystallized the enzyme from E. coli K 12 and Proteus mirabilis F-lac+ (72). Craven et al. (68) developed a fractionation procedure which does not include the esthetically more satisfying crystallization but yields large quantities of enzyme that is free from contamination with minor components. A similar procedure was used by Steers and Shifrin for isolating an inactive mutant protein from E. coli K 12 (73 ) . This method is very convenient and has been used in our laboratory with a slight modification of the original procedure.

The enzyme, after the DEAE-Sephadex ste3, may be crystallized and recrystallized as described by Wallenfels and Malhotra ( 2 ) . The first crystals are thin, hexagonal plates. On recrystallization, hexagonal needles are obtained. The crystallized produat has a turnover number of 3.58 X lo5 moles of o-nitrophenyl P-D-galactoeide per minute per mole of enzyme a t 25°C in 5 X 10-2M sodium phosphate buffer (pH 6.8), containing 10-3M MgCl, and 2.6 X

Recently, Tomino and Paigen (74) introduced an interesting technique for isolating the enzyme by affinity chromatography. They used the competitive inhibitor p-aminophenyl P-D-thiogalactoside coupled to cross- linked y-globulin as a specific chromatographic adsorbent and obtained the enzyme in 99% purity from crude extracts in a single column opera- tion, although only in milligram quantities. This “functional purification” was further developed by Steers e t al. (75) , who used the same thiogalactoside covalently attached to agarose derivatives and obtained the enzyme from 25 g bacteria in 95% yield.

M substrate.

B. P-GALACTOSIDASES FROM OTHER MICROORGANISMS

Purification procedures regarding p-galactostdases from microorganisms other than E. coli have been reported. The eizymes from Streptococcus

71. D. Perrin, Ph.D. Thesis, Paris (1965). 72. C. Colby and A. S. L. Hu, BBA 157, 167 (196t;). 73. E. Steers and S. Shifrin, BBA 133, 454 (1967). 74. S. Tomino and K. Paigen, in “The Lactose Operon” JJ. R. Beckwith and D.

75. E. Steers, P. Cuatrecasas, and H. R. Pollard, JBC 246, 196 (1971). Zipser, eds.), p. 233. Cold Spring Harbor Monographs, 1970.


lactis (76) , Aeromonas formicans (77) , Saccharom yces lactis ( d l ) , Sporobolomyces singularis (78) , Aerobacter cloacae (79) and the 6- phospho-P-galactosidase from Staphylococcus aureus (80) have been isolated in a state of high purity, as judged by several physical procedures. Partial purification has been achieved for p-galactosidase from Shigella dysenteriae (81 ) , Paracolobacterium aerogenoides (82), Saccharomyces fragilis (&I) , Bacillus subtilis (15) , Bacillus megater- ium (83) , Diplococcus pneumoniae (12) , and Tricholnonas foetus (22).

C. ANIMA~ p-GALACTOSIDASES

Two classes of animal p-galactosidase have received special attention because of their physiological importance : the enzymes attacking complex galactosylsphingolipides that were purified from brain and intestine, and the intestinal lactases because of their involvement in hydrolysis of dietary disaccharides.

Bowen and Radin described the 300-fold purification of a lysosomal /3-galactosidase that acts on the brain lipid ceramide galactoside from rat brain (37). A less purif?ed preparation from rat and calf brain, which exhibits similar properties, was obtained by Gatt and Rapport (84). Jungalwala and Robins (85) described the purification of two p-galactosidases from rabbit brain that catalyze the hydrolysis of lactose and synthetic substrates but are inactive toward galactosyl cerarnides.

The first highly purified p-galactosidase from intestine was prepared by Wallenfels and Fischer (86). They achieved a 2000-fold purification of the enzyme from calf intestine. However, the preparation contained rather large amounts of carbohydrates and was not completely pure as shown by ultracentrifugation ( 2 ) . Alpers (87) isolated two enzymes from rat intestine, a lysosomal p-galactosidase and a lactase located in the brush border, which were purified 1000-fold. Small intestinal p-galactosidase from monkey was purified to a high degree by Swamina-

76. G. A. McFeters, W. E. Sandine, and P. R. Elliker, J. Bacteriol. 93, 914 (1967). 77. S. R. Rohlfing and I. P. Crawford, J. Bacteriol. 91, 1085 (1966). 78. J. A. Blakely and S. L. MacKenzie, Can. J. Biochem. 47, 1021 (1969). 79. R. P. Erickson and E. Steers, ABB 137, 399 (1970). 80. W. Hengstenberg, W. K. Penberthy, and M. 1,. Mom, Birropeon J . Biochem.

81. S . Sarkar, J. Bacteriol. 91, 1477 (1966). 82. J . M. Anderson and H. V. Rickenberg, J. Bacteriol. 80, 297 (1960). 83. S. R. Rohlfing and I. P. Crawford, J. Bacteriol. 92, 1258 (1966). 84. S. Gatt and M. M. Rapport, BBA 113, 567 (1966). 85. F. B. Jungalwala and E. Robins, JBC 243, 4258 (1968). 86. K. Wallenfels and J. Fischer, 2. Physiol. Chem. 321, 223 (1960). 87. D. H. Alpers, JBC 244, 1238 (1969).

14, 27 (1970).

20. p-GALACTOSIDASE 627

than and Radhakrishnan (88). Partial purification of small intestine p-galactosidase from man has been reported by Semenza et al. (89) and from rat by Asp and Dahlqvist (90), Asp (91) , and Doell e t al. (92) . Two intestinal enzymes from the rat were purified by Brady et al.: One catalyzes the hydrolysis of both galactosyl and glucosyl ceramide (39) , and the other is a special trihexosidase which is only active toward the terminal galactosyl residue from trihexyl ceramide (4U).

Isolation of /3-galactosidase from ram testis was reported by Caygill et al. (93) and from sheep thyroid gland by Chabaud et al. (94) . In general, animal /3-galactosidases have been obtained in a less purified state than the microbial enzymes. One notable exception is two p-galactosidases from the digestive juice of the snail Helix pomatia, which were purified to apparent homogeneity (95) .

V. Physicochemical Properties

The properties of crystallized /3-galactosidase werc examined by ultracentrifugation (68, 96), analytical gel electrophoresis and moving- boundary electrophoresis (68), electrofucusing (97) , and Ouchterlony double diffusion test (73) . The results indicated that the enzyme preparations were homogeneous in respect to size, shape, net charge, and immunological behavior. The isoelectric poinl;, found by electrofocusing, is 4.61 (97) .

A. SIZE, SHAPE, AND QUATERNARY STRUCTURE

In the ultracentrifuge, a monodisperse component can bc observed which is characterized by a sedimentation coefficient, corrected to zero protein concentration and water a t 20°, of 16s (96 ) , 16.4s (73), or 15.9s (77) . Sund and Weber (96) deteymined all relevant hydro-

88. N. Swaminathan and A. N. Radhakrishnan, rnd. J . Biochem. 6 , 101 (1969). 89. G. Semenza, S. Auricchio, and A. Rubino, BBA 96, 487 (1955). 90. N. G. Asp and A . Dahlqvist, BJ 106, 841 (1968). 91. N. G. Asp. BJ 117, 369 (1970). 92. R. G. Doell, G. Roscn, and N. Kretchmer, 1 ~ 1 . o ~ . N u t . Acnd. Sci. U . S . 54,

93. J . C. Caygill, C. P. J . Roston, and F. R. Jevons. BJ 98, 405 (1966). 94. 0. Chabaud, 6. Bouchilloux, and M. Ferrand, BBA 227, 154 (1971). 95. R. Got and A. Marnay, Etiropenn J . Biochein. 4, 240 (1968). 96. H. Sund and K. Weber, Riochem. Z. 337, 24 (1963). 97. F. G. Loonticns, impuhlishcd results (1968).

1268 (1965).


TABLE I

E. wli ML 35 AND ML 309 MOLECULAR CONSTANTS OF fl-GAL.4CTOSIDASE FROM

Constant ML 35 MI, 309

16.14 S 0.146 3 . 1 3 F

521,000

0 .73 1 .26 3 .78 4 .97

2.09 X lo6 5 . 7 5 . 4

-

15.93 S 0.149 3 . 1 2 F

516,000 0 . 7 6 0 .73 1 .27

- 5 . 9

0 Partial specific volume calculated from the amino acid composition.

dynamic data of the enzyme from E. coli MI, 35 and ML 309 (Table I ) . These data indicate that p-galactosidase has a molecular weight of 518,000. Its shape can be described as an oblate ellipsoid of rotation with an axial ratio of about three assuming 30% hydration. The length and height of the molecule were calculated to be 150 and 50A, respectively (98). Craven et al. (68) studied the molecular weight by low and high-speed equilibrium sedimentation and found values of 489,000 and 538,000, respectively, whereas Colby and Hu (72) by using the same method reported molecular weights of 503,000 and 491,000. Since the enzyme preparation used by Sund and Weber (96) contained small amounts of heavier material the molecular weight of P-galactosidase may actually be close to 500,000.

/I-Galactosidase is dissociated into smaller subunits by a variety of methods such as treatment with urea and guanidine hydrochloride (99), succinylation (IOO), pH > 11.5 or < 3.5 ( 5 I ) , and treatment with p - mercuribenzoate or other mercurials (99, 101). Since the urea-treated enzyme can spontaneously resume its native conformation after removal of the denaturant, it is possible to obtain hybrids of enzyme labeled with heavy isotopes (*T and lW) and light isotopes ('T and "N) and to

98. H. Sund and K. Weber, Airgezci. Chem. Znt. Ed. 5, 231 (1966). 99. I<. Wallenfels, H. Sund, and K. Weber, Biochem. Z. 338, 714 (1963). 100. K. Wrher, Ph.D. Thesis, Frciburg (1964). 101. F. G. Loontiens, K. Wallenfels, and R. Weil, Europenn J. Biochem. 14,

138 (1970).


compare the densities. Using this method, Zipser (102) could establish that the native enzyme is composed of four cionomers. This result is in agreement with findings of Wallenfels et al. (99) who investigated the dissociation of reduced and carboxymethylated enzyme in urea and guanidine hydrochloride. I n 5 M guanidine hydrochloride the protein yields a single component with an s,O,,, of 3.65 S and Die,, of 2.5 X l W 7 cm2 sec-'. The molecular weight calculated from these data is 147,000. This value was confirmed later by Shifrin and Steers (103) who obtained a molecular weight of 118,000 by sedimentation equilibrium in 8 M urea. Ullmann e t al. (104) studied the molecular weight in 6 M guanidine hydrochloride by the Archibald method and found a value of 135,000.

The tetrameric structure of the enzyme wa;j directly demonstrated by electron microscope studies which indicated furthermore that the four subunits are arranged a t the corners of a square (105) (Fig. 1). The dimensions of the molecule determined by t,iis method are about 120 and 7 0 A which agrees closely with the values obtained from hydro- dynamic data. Additional, although less convincing, evidence for the tetrameric structure comes from chemical studies of the enzyme, especially from end group determinations as shown in the following section. However, the question of whether the monomer itself is the smallest physical subunit or is composed of smaller polypeptide chains is far more difficult to answer.

Two groups of workers have reported that relatively mild treatment of the native molecule breaks P-galactosidase into material with a molecular weight significantly smaller than 130,000. Wallenfels et al. (99) observed that on oxidation with performic acid the molecular weight decreases to about 35,000. In 0.1% SDS, the nio!ecular weight was found to be about 60,000. Dissociation of the enzyme into smaller units having a molecular weight of 46,000 also appears to o1:cur in a mixture of formic and acetic acid (106). Steers e t al. have reported similar molecular weights for enzyme treated with 70% formic acid, 0.5% SDS and 0.05M NaOH (107).

102. D. Zipser, J M B 7, 113 (1063). 103. S. Shifrin and E. Steers, BBA 133, 463 (1967). 104. A. Ullmann, M. E. Goldberg, D. Perrin, and J. Monod, Biochemisliy 7,

105. U. Karlsson, S. Koorajian, I. Zabjn, F. S. Sjostrand, and A. Miller, J. Ultra-

106. K. Weber, H. Sund, and K. Wallenfels, BiooLem. 2, 339, 498 (1964). 107. E. Steers, G. R. Craven, C. B. Anfinsen, and J. 1,. Brthune, JBC 240,

261 (1968).

struct. Res. 10, 457 (1964).

2478 (1965).

630 I(. WALLENFELS AND R. WEIL

FIQ. 1. For.maldehyde-treated P-galactosidase from E. coli after staining with uranyl acetate. 250,OOOX. From Karlsson et al. (105) (by courtesy of Dr. I. Zabin).

Complementation studies have demonstrated polypeptide products of the z gene that are smaller than 130,000 (108,109). Such complementation tests between strains carrying various point mutations in z, and a series of mutants with deletions extending various distances into z , allowed the gene to be divided into three regions a, p, and o according to their distance from the operator. The a and o peptide have been purified from several deletion mutants and have been found to possess a molecular weight of one-fourth and one-third, respectively, of the monomer. Taken together, these observations suggested that a, p, and o might correspond to different cistrons within the lac operon and might be identical to the smaller units of molecular weight 35,000-60,000. The structure of the enzyme thus appeared to be rather complex, each monomer consisting of three noncovalently associated polypeptide chains.

Newton (110), however, investigating the polar effect of nonsense mutations in the lac operon; concluded that z does not contain inter- cistronic punctuations like that represented by the z-y or the y-a punc- tuation. Hence, z should represent one single cistron and the monomer of 130,000 molecular weight should be synthesized as a single chain. More-

108. A. Ullmann, D. Perrin, F. Jacob, and J. Monod, J M B 12, 918 (1965) 109. A. Ullmann, F. Jacob, and J. Monod, J M B 24, 339 (1967). 110. A. Newton, Cold Spring Harbor Symp. Quant. Biol. 31, 181 (1966).

20. P-GALACTOSIDASE 63 1

over, Ullmann e t al. (111) presented results indicating that tlic (Y and w

peptides do not exist as such in wild type P-galactosidase, whereas these units are easily recovered froin enzyme formed by coiiil)leiiiciitation.

This discrcpancy betwceii physical mcasureiiicnts and genetic studics prompted Weil et al. (112) to reinvestigate the dissociation of P-galactosidase in formic acid-acetic acid mixtures. They established that tlie sedimentation coefficient in this solvent does lot cxhibit the usual linear concentration dependence but incrcascs considcrably a t low protein concentrations because of a large polyclectrolyte effect in the strongly protonizing solvent. The addition of 0.2 iM KCl permits tlic unambiguous determination of thc molecular weight, which is found to bc 128,000. The same molecular weight was incasurcd by light hcattcriiig iii tlie prcscnce of 0 .2M sodium formate or by performing the measurements a t very low protein concentrations wlicre thc polyclcc trolytc cffcct is cliininatcd. I n addition, Weber and Osborn (113) demonstrated by clcctrophoresis in SDS acrylamide gels that in SDS p-galactosidase possesses a molecular weight larger than that of paramyosin (molecular weight 100,000). Since, in the presence of SDS, proteins bind large amounts of the detergent and should therefore carry a considerablc negative charge, i t is conceivable that the appearance of material with apparent molecular weight in thc raiigc of 40,000-60,000, as determined in the ultracentrifuge, is also the result of a polyelcctrolytc cffcct that is not compensated for by the addition of counterions. In conclusion, i t can be stated that the smallest physical subunit, of P-gnlactosidasc has a molecular weight in the range of 130,000 and corresponds to the entire z gene.

Some enzymically inactive P-galactosidasc proteins were purified to a state of homogeneity sufficient to investigate their size and quaternary structure. Perriii (71) isolated three mutant proteins (Czp, Cz,, and C Z , ~ ~ ) and found the molecular weight of Czp to be similar to that of wild type P-galactosidase whereas the molecular weight of CzliS (104,OOO) corresponds to that of the monomer. The protein Czl was characterized by Steers and Shifrin (73) and was shown to lie a dimer with a molecular weight of 247,000. Thus, the mutation producing Cz, gives rise to a monomer fully capable of forming a dimer but not capable of forming a tetramer, and the mutation of CzliS also affects the ability of the monomer to form the dimer. On the other hand, the tetramer Czp seems to be less stable than wild type galactosidase, dissociating spon-

111. A. Ullmann, F. Jacob, and J. Monod, JMLl 32, 1 (1968). 112. R. Weil, W. Burchard, and W. Wallenfels, unpublished results (1969). 113. K. Weber and M. Osborn, JBC 244, 4406 (1969).


taneously into material corresponding to the dirner and monomer (71). The three mutant proteins possess very low levels of enzymic activity

which in the case of Cz, and C Z , ~ ~ is due to the presence of small amounts of mutant tetramer. This finding suggests that the tetrameric structure is a prerequisite for enzyme function, as is shown by the fact that active enzyme obtained by complementation of Cz, with Czli8 has the sedimentation coefficient of 1 6 s characteristic of wild type enzyme (114).

"-Complemented P-galactosidase was shown by Goldberg (115) to be a tetramer with a molecular weight of 595,000, thus heavier than the wild type enzyme. The monomer with a molecular weight of 150,000, is built up of two nonidentical polypeptide chains, corresponding to the acceptor and to the w chain with molecular weights of 110,000 and 4O,OOO, respectively (116). The excess molecular weight could be attributed to a redundant polypeptide fragment present in both the acceptor and the o peptide, which can be easily removed by treatment of the complemented enzyme with papain (115).

Brown et al. (117) and Berg et al. (118) isolated the incomplete polypeptide chains from an ochre and an amber mutant strain in almost pure form. The molecular weight of the mutant proteins was found to be 122,000 (ochre) and 89,000 (amber), which is in excellent agreement with the size predicted from the position of the mutation.

Data on physicochemical properties of P-galactosidase from origins other than E. coli are surprisingly scant. The enzymes from Aero- monas formicans and Aerobacter cloacae appear to have gross physicochemical properties similar to that of E . coli (77, 79). The 6- phospho-p-galactosidase from Staphylococcus aweus has a molecular weight of 50,000 and consists of one polypeptide chain (80). The molecular weights of the enzymes from Bacillus megateriuin (83) and Sporobolomyces singularis (78) were found to be in the range of 140,000- 150,000. The molecular weight of two highly purified enzymes from Helix pomatia can be estimated from their sedimentation coefficients (10 and 6.8 S) to be about 250,000 and 125,000 (95).

Most physicochemical studies on mammalian p-galactosidase were performed on crude preparations. Several investigators found molecular weights of 80,000 for lysosomal enzymes from rabbit brain (85), rat kidney (M), and ox liver (119). I n addition, an enzyme fraction of

114. D. Perrin, Cold Spring Harbor Symp. Quant. Biol. 28, 529 (1963). 115. M. E. Goldberg, JMB 46, 441 (1969). 116. M. E. Goldberg and S. J. Edelstein, JMB 46, 431 (1969). 117. J. L. Brown, D. M. Brown, and I. Zabin, Proc. Nut. Acad. Sci. U. S. 58,

118. A. P. Berg, A. V. Fowler, and I. Zabin, J . Bacteriol. 101, 438 (1970). 119. F. Chytil, BBRC 19, 630 (1965).

1139 (1967).


40,000 molecular weight possessing both j3-galac tosidase and P-glucosidase activity was shown to occur in rat kidney and ox liver. Enzymes having larger molecular weights [103,000 (85) and 127,000 (119)] were also found. However, as Asp was able to demonswate in the case of lyso- soma1 p-galactosidase from rat intestine (120) , elution patterns of crude enzyme preparations depend largely on ionic strength and pH of the elution buffer. Therefore, calculation of molecular weights based on gel chromatography data alone may be misleading.

B. ASSOCIATED FORMS OF P-GALACTOSIDASE

Aggregated material with sedimentation coefficients of 23, 27, and 36 S was found by Wallenfels et al. (66) in prep:trations of crystallized P- galactosidase. Later, Sund and Weber (96) demonstrated that the amount of 2 3 s component increases considei:ably when the pure 1 6 s fraction stands in the absence of SH compounds. More recently, Reithel et al. (121) pointed out that the use of thioglycollic acid in their method of purification reduces large aggregates to :i minimum. Rohlfing and Crawford (77) were able to isolate a 23 S component with a considerably reduced specific activity, when compared with the 16 S component. Appel et al. (50) reported that crude extracts of E . coli contain as many as seven active forms of P-galactosidase, as shown by electrophoresis on polyacrylamide gels and sucrose gradient centrifugation. Marchesi et al. (51) purified a mixture containing various multiple forms of the enzyme with sedimentation values of 23, 27, 32, 36, and 41-4553, which they considered to be multiples of 6, 8, 10, 12, and 16 monomeric units, respectively. These aggregates appear to be more sensitive to denaturation by heat and urea and to possess slightly different net charges than the tetramer as judged by ion exchange chromatography. On the other hand, their enzymic properties, such as specific activity and pH dependence, are not different from that of the tetramer (61). Since they are presumably aggregates of the smallest enzymically active form, the tetramer, they can probably be considered as associated forms, not as isoenzymes.

The exact conditions which cause the tetramer to aggregate are still unknown. However, i t is interesting to note that the mutation producing Cap, which affects tetramer formation, also prevents formation of the associated forms (71). In addition, comparative ” studies of enzymes

120. N. G. Asp, BJ 117, 369 (1970). 121. F. J, Reithel, R. M. Newton, and M. Eagleson, Nature 210, 1265 (1966).


from several bacterial species, and of a modified P-galactosidase from E. coli containing selenomethionine instead of methionine, showed a direct correlation between formation of aggregates and the stability of the tetramer against heat and urea denaturation (122) .

C. DENATURATION AND RENATURATION STUDIES

1. Heat Denaturation

P-Galactosidase from E. coli is fairly heat stable in the presence of mercaptoethanol. With 0.1 M mercaptoethanol and 0.01 MgC1, a 50% loss in activity is observed after 40 min a t 55”, whereas without mercaptoethanol all activity is lost after 10 min a t this temperature (68). Study- ing thermal denaturation of the enzyme a t the single molecule level, Rotman (59) established that molecules in a heat-inactivated population are either fully active or completely inactive. This is in contrast to the inactivation caused by prolonged storage which results in partial loss of activity of individual molecules (123) . Gest and Mandelstam (124) reported that the enzyme becomes remarkably heat labile in the presence of fructose 1,6-diphosphate. Heat stability is also considerably affected by various metabolic intermediates derived from glucose metabolism, such as fructose, fructose 6-phosphate, ribose 5-phosphate, and DPNH (125) . The significance of these findings, which were originally thought to be related to the phenomenon of catabolite repression, is not clear.

Melchers and hlesser (126) investigated the heat stability of wild type P-galactosidase and of several antibody activatable mutant proteins in the presence of specific antibody. They reported that the wild type enzyme and all but one activatable mutant protein exhibit an increased stability against thermal inactivation when complexed with antibodies directed against wild type P-galactosidase.

Active enzyme obtained by complementation of point mutants with a or o peptide is very sensitive to heat denaturation (111) . Similar findings have been reported for the mutant enzyme Czp (71 ) . Langridge (127) studied the heat stability of a series of amber mutants suppressed by the sup D suppressor gene. Since this suppressor causes the insertion of

122. R. P. Erickson and E. Steers, J. BucterioZ. 102, 79 (1970). 123. B. Rotman, in “The Lactose Operon” (J. R. Beckwith and D. Zipscr, eds.),

124. H. Gest and J . Mandelstam, Nature 211, 72 (1966). 125. M. E. Brewer and V. Moses, Nature 214, 272 (1967). 126. F. Melchers and W. Messer, BBRC 40, 570 (1970). 127. J. Langridge, J. Bncteriol. 96, 1711 (1968).

p. 279. Cold Spring Harbor Monographs, 1970.


serine a t the point in the polypeptide chain o1,herwise terminated a t the amber triplet, the resulting enzymes differ from each other only in the position of substitution of serine for the original amino acid. Langridge could establish that insertion of serine a t the carboxyl-terminal third of the polypeptide chain causes pronounced thermal instability, whereas insertion a t the first two-thirds gives an irregular alternating pattern of heat sensitivity. Similar results were oLtained by using enzyme from glutamine-suppressed amber mutants. Increased heat sensitivity was also observed when the unphysiological amino acids, selenomethionine (128) and thienylalanine (129), were incorpoi-ated into p-galactosidase.

2. Influence of p H

There is no loss in enzymic activity when a solution of p-galactosidase is maintained for 30 min a t 40" and pH 6-8. The stability decreases sharply below pH 6 and slowly as the pH is raised above 8 (1, 2 ) . At pH 3.5 and 11.5 the enzyme is completely dissociated into inactive monomers (51) .

3. Urea Studies

Shifrin and Steers (103) have studied the effect of increasing concentrations of urea on the dissociation of P-galactosidase as reflected by the sedimentation velocity, enzymic activity, and protein fluorescence. They found that the tetramer is completely dissociated into inactive monomers in 6 M urea with no intermediate forms sufficiently stable to be detectable in the ultracentrifuge. However, they did not establish a direct relationship between inactivation and dissociation. Wickson and Huber (130) demonstrated that loss of activity and loss of tetrameric structure are not parallel reactions and suggested the existence of an inactive form of tetramer in 6 &I urea. Lactose was reported to stabilize both the active and the inactive tetramer.

4. Renaturation Studies

Zipser (102) was the first to find that active enzyme could be recovered in good yield after treatment with 8iM urea. Perrin and Monod (131) later showed that fairly good recovery could be obtained also after treatment by heat or 6 M guanidine hydrochloride. Weber et al.

128. R. E. Huber and R. S. Criddle, BBA 141, 587 (1967). 129. J. Jana6ek and H. V. Rickenberg, BBA 81, 108 (1964) 130. V. M. Wickson and R. E. Huber, BBA 207, 150 (1970). 131. D. Pcrrin and J. Monod, BBRC 12, 425 (19B3).

636 K. WALLENFELS AND R. WELL

(106) obtained partial reactivation of enzyme treated with a mixture of formic and acetic acid. However, correct refolding of both heat- and acid-inactivated enzyme occurs only after dissolving the denaturated protein in 8 M urea. Thus, i t is not surprising that alkali dissociated enzyme does not reassociate spontaneously when the pH is returned to 7.5 (51).

Ullmann and Monod (132) carefully investigated the renaturation of urea-treated enzyme. They established that renaturation by dialysis in the absence of Mg2+ ions yields tetrameric enzyme which they found, however, to be virtually inactive. Activity was instantaneously restored upon addition of Mg2+. The latter observation is very surprising since Mg2+ was shown not to be essential for binding and splitting of substrate although it increases the rate of hydrolysis considerably (133, 134). Removal of urea by dialysis in the presence of Mg2+ resulted in formation of large inactive precipitates, and the authors concluded that during removal of the denaturant divalent cations favor interactions between the unfolded polypeptide chains, thus leading to water insoluble aggregates. This interpretation is supported by the fact that renaturation from guanidine hydrochloride solutions, which usually yields only low recoveries of activity, is quite successful a t low protein concentrations, i.e., under conditions where intermolecular interactions are not favored.

VI. Chemical Properties

Crystalline P-galactosidase from E. coli ML 309 contains 51.5% carbon, 6.3% hydrogen, 16.1% nitrogen, and 0.93% sulfur, but no carbohydrate or phosphate (66). The amino acid composition of P-galactosidase from different strains of E. coli was determined in several laboratories. The results are given in Table I1 together with data obtained from amino acid analyses of enzymes from the organisms Aeroinonas formicans (77), Aerobacter cloacae (79) , and Sporobolonzyces singularis (78). Examination of these data indicates that the analyses of proteins from strains M L 35 and RIL 309 agree fairly well with each other. There are more dissimilarities between MI, and K 12 proteins. However, it is likely that these variations result from technical reasons rather than differences in chemical composition, thus reflecting the progress in amino acid analysis. The data presented by Huher and Criddle for the

132. A. Ullmann and J. Monod, BBRC 35, 35 (1969). 133. F. J. Reithel and J. C. Kim, ABB 90, 271 (1960). 134. K. Wallenfels and F. Kubowitz, unpublished results (1966).


TABLE I1 AMINO ACID COMPOSITION OF ~-GALACTOSIDALE (MOLES OF AMINO

ACID PER lo6 g O F PROTEIV)

Amino E. coli E. coli E . coli E. coli acid ML 3Oga ML 35a K 12b K 12c A. formiccznsd A. cloacae8 S. singularisf

LYS His -4% g-cys ASP Thr Ser Gl~i Pro

Ala Val Met Ile Leu TYr Phe TrP

GlY

19.0 31.2 56.5 17.1 88.6 45.9 47.6 95.7 44.1 60.6 65.0 53.0 20.1 36.2 82.1 20.8 31.8 35.0

18.3 32.5 51.2 17.5 83.6 46.0 56.0 93.0 47.1 60.9 61.5 49.4 20.5 32.3 72.6 24.4 30.1 35.5

21.5 26.7 54.8 14.1 91.3 48.2 49.7

105.5 49.5 63.1 69.0 55.6 17.8 35.6 81.6 26.6 33.4 26.0

20.0 13.3 26.7 33.2 54.0 68.0 12.7 16.8 90.5 80.0 51.9 37.4 51.3 31.1

108.0 90.4 52.5 60.3 62.5 70. I 69.6 86.5 55.6 49.4 15.6 23.9 31.6 28.1

25.1 24.4, 33.4 27.8 26.0 41.0

83.0 102.4:

12.4 28.6 64.6 21.7 97.5 47.7 56.0 91.5 50.5 65.0 72.6 49.6 11.6 38.4 90.5 18.1 32.0 21.1

44.4 18.6 26.4 0

88.5 51.5 53.6 77.8 44.3 83.6 80.8 44.4 0

36.4 61.5 13.6 40.6 Q

Data from Wallenfels et al. (155). Data from Craven et al. (68).

Data from Rohlfing and Crawford (77). Data from Erickson and Steers (79).

f Data from Blakely and MacKeneie (78). 9 Not determined.

0 Data from Berg et a2. (118).

enzyme from strain K 12 may be questioncd because they agree so little with the data obtained by other laboratories (128).

The enzymes from Aerobacter cloacae and A wornonas formicuns, which are very similar in their physical and enzymic properties to the enzyme from 8. coli, have a similar amino acid composition. In contrast, the enzyme from the yeast Sporoboloinyces singularis, which is compIetely unrelated to the enteric bacteria, displays a strikingly different amino acid composition. The lack of the sulfur-containing amino acids is especially noteworthy. A special feature of the E. coli enzyme is its high content of aromatic amino acids, especially of tryptophan, which accounts for the extinction coefficient of 1.92 cm2/mg 116'6) or 2.09 cm2/mg (68).

Very few data are available concerning the chemistry of animal P-galactosidases. So far, the most purified preparations were found

135. K. Wallenfels, C. Streffer, and C. Golker, Bwchem. Z. 342, 495 (1965).


to contain large amounts of carbohydrates (86, 95) , up to 88% in the case of the enzyme from calf intestine. The biological significance of this extremely high carbohydrate content is not clear. Attempts to separate the carbohydrate moiety from the protein resulted in complete inactivation ( 2 ) . Lysosomal P-galactosidases seem to contain N-acetyl neur- aminic acid. Treatment of enzyme preparations with neuraminidase changes the electrophoretic behavior by shifting the isoelectric point to a higher pH (136).

Much structural work on P-galactosidase from E . coli has been performed to aid in elucidating the quaternary structure of the enzyme and to decide whether the monomer is composed of one or more than one polypeptide chain.

The minimum chemical molecular weight has been found to be one- fourth that of the complete protein. This conclusion is based on results obtained after cleavage of the protein with trypsin and cyanogen bromide. The enzyme contains approximately 500 residues of arginine + lysine and 96 residues of methionine. Approximately 86 ninhydrin-positive peptides could be detected on peptide maps of tryptic digests ( lor ) , and 20-25 peptides were found by gel electrophoresis (107) or amino terminal analysis (137) after treatment with cyanogen bromide. Thus, i t appears that the four monomers are chemically identical and represent a unique sequence of amino acids.

Determination of end groups yielded less clear-cut results. The presence of threonine as an amino-terminal residue was first observed by Cohn and confirmed by several groups (138, 107, 139). Careful amino- terminal analysis by the Stark and Smyth carbamylation method yielded 1 mole of threonine per monomer of 135,000 molecular weight (140). Furthermore, when dinitrophenylated enzyme was treated with cyanogen bromide, the dipeptide DNP-threonine-homoserine could be isolated, indicating an amino-terminal sequence NH,-threonine-methionine (137).

Attempts to detect blocked amino groups, especially N-acetyl groups, gave negative results (140). Carboxyl-terminal studies using hydrazinol- ysis or carboxypeptidase B showed the presence of 1 mole of lysine per monomer (140-142). I n addition, the carboxypeptidase experiments suggested the possible sequence Tyr-Glu NH,-Lys COOH (140).

136. A. Goldstone, P. Konecny, and H. Koenig, FEBS Letters 13, 68 (1971). 137. J. Katze, S. Shridhara, and I. Zabin, JBC 241, 5341 (1966). 138. J. Monod, Angew. Chem. 71, 685 (1959). 139. K. Wallenfels and A. Arens, Biochem. 2. 333, 395 (1960). 140. J. L. Brown, S. Koorajian, J. Katze, and I. Zabin, JBC 241, 2826 (1966). 141. S. Koorajian and I. Zabin, BBRC IS, 384 (1965). 142. S. G. Korenman, G. R. Craven, and C. B. Anfinsen, BBA 124, 160 (1966).


Taken all together, these findings seem to he in line with the results from physical studies that the monomer of 135,000 molecular weight represents one polypeptide chain with threonino and lysine as the amino- and carboxyl-terminal amino acid. There have been, however, several observations froin end group studies which are not consistent with this conclusion. Erickson and Steers reported the presence of pyrrolidonc carboxylic acid in peptides obtained after pronase digestion and ion exchange chromatography (143). Wallenfels and co-workers (139, 144) found glutamic acid in addition to threoiiine as amino-terminal residues. In addition, when performic acid oxidized /3-galactosidase was treated with leucine aminopeptidase, considerable amounts of various amino acids such as methionine, alanine, serinc, and glutamic acid could be detected (144). However, these findings could be artifacts. Pyrrolidone carboxylic acid can be formed by cyclization of internal glutamine residues which become amino-terminal after proiiase treatment, and small amounts of proteolytic contaminants could account for the results of leucine aminopeptidase treatment. In addition, it was shown by Givol e t al. that exposure of /I-galactosidnsc to trypsin and papain results in multiple cleavages of peptide bonds, nlthougli the fragmented enzyme retains essentially native enzymic activity arid sedimentation behavior (145). Thus, a protease present in a particular /3-galactosidase preparation could split a few sensitive peptide bonds and lead to new end groups. I n fact, i t could be shown that even highly purified P-galactosidase contains traces of proteolytic activity (146).

Recently, the first results of sequence analysis were published (147). Sixty tryptic peptides were isolated in pure form and analyzed. The complete primary structure may be known i i the near future.

VII. Immunological Properties

P-galactosidase from E. coli is an excellmt immunogen giving rise to precipitating antibodies which do not inhibit its enzymic activity (148). Many z-mutant strains produce proteins that are enzymically

143. R. P. Erickson and E . Steers, BBRC 37, 736 (1969). 144. K. Wallenfels and C. Golker, Biochem. 2. 346, 1 (1966). 145. D. Givol, G. R. Craven, E. Steers, and C. B. Anfinsen, BBA 113, 120 (1966). 146. K. Wallenfels and G. Laule, unpublished results (1971). 147. A. V. Fowler and I. Zabin, JBC 245, 5032 (1970). 148. M. Cohn and A. M. Torriani, J . Immunol. '89, 471 (1952).


inactive, but cross-react with antibody to the wild type (149). Using the antigenic activity to anti-p-galactosidase as an assay, Zabin and co- workers were able to isolate several mutant proteins which correspond to incomplete polypeptide chains (117, 118). Melchers and Messer prepared an immunoadsorbent from antiserum against wild type p-galactosidase coupled to sepharose and were able to isolate a series of inactive, but antibody activatable, mutant proteins in high yield (150).

Fowler and Zabin obtained interesting information about the antigenic determinants of p-galactosidase by analyzing the immunological activity of several nonsense, missense, and deletion mutant proteins (151). Prematurely terminated chains from nonsense mutants could be grouped into two general classes, those of molecular weight 50,000-70,000 and those of molecular weight 90,000-120,000. Within each class, the polypeptide chains seem to possess identical immunogenic sites, but the chains of the two classes are different from each other. The existence of the two classes suggests that each has a conformation somewhat different from the other but related to that of the native protein. Deletion mutants belong to one or the other of the two classes, according to the position of the deletion.

Rotman and Celada have isolated a cross-reacting protein from a z- mutant which was activated 550-fold by antiserum to the wild type p- galactosidase (152). The molecular weight of the mutant protein was found to be 495,000, indicating that it is a tetramer. The activation by antibodies is therefore likely to occur via a conformational change resulting from the interaction of the antigenic sites with the binding sites of the antibody molecule. This activation does not require divalent antibodies, since Fab fragments of antibodies are effective as well (153). Interestingly enough, activation does not take place when antiserum directed against the mutant protein itself is used (153).

I n related studies, Messer and Melchers investigated the mapping of eleven antibody activatable mutants (154, 155). At least two groups of mutations could be distinguished, and each group sccms to be activated by a different antibody population.

149. D. Perrin, Ann. N . Y . Acnd. Sci. 103, 1058 (1963). 150. F. Melchrrs and W. Messn., Europenn J . Biochein. 17, 267 (1970). 151. A. V. Fowlcr and I. Zabin, J M B 33, 35 (1968). 152. M. B. Rotman and F. Celada. Proc. Nnt. Acnd. Sci. U . S . 60, 660 (1968). 153. F. Cclada. R. Strom, and K. Bodlnnd, in “Thr Lactose Operon” (J. R .

Beckwith and D. Zipser, eds.), p. 291. Cold Spring Harbor Monographs. 1970. 154. W. Messer and F. Melchers, in “The Lactose Opcron” (J. R. Beckwith and

D. Zipser, eds.), p. 305. Cold Spring Harbor Monographs, 1970. 155. W. Mrsser and F. Melchcrs, Molec. Gen. Genetics 109, 152 (1970).


p-Galactosidase from E. coli is weakly cross-reactive to P-galactosidase from Aerobacter cloacae. This immunological relationship was used to purify the A. cloacae enzyme on an immunoadsorbent containing antibody to the E. coli enzyme (79). Antisera were prepared against P-galactosidase from Helix pomatia, from ra t intestine, snd from various mouse tissues (53, 92, 156). None of the antisera inactivates the corresponding enzyme. The antiserum to intestinal P-galactostdase was used for histochemical localization of the enzyme in the brush border of jejunal cells (9%').

VIII. Enzymic Properties

Studies on the enzymic properties of P-galactosidase have been greatly facilitated by the fact that a large number of easily prepared or naturally occurring substrates are available. Of all the p-galactosidases so far investigated, that from E . coli has been the most thoroughly characterized so that it is tempting to use this enzyme tis a standard reference in studying P-galactosidases from other sources.

A. SPECIFICITY

As is the case with many glycosidases, the sliecificity of the enzyme is confined to the sugar moiety and to the anomeric character of the linkage but not to a particular aglycon. Based on the simple Michaelis- Menten pathway

k i koat

k-i E + SeES-- , E + P

where P stands for product and k - , / k , equals tl-ie dissociation constant I<, of the enzyme-substrate complex, the kinetic parameters K , = (k , , , + k - , ) / k , and V , = k,,,[E] have been derived for a large number of substrates. Assuming that for most substrates k - , >> lc,,, and hence K , =

Ks = lc-Jcl, the experimentally determined value of K,, is a measure of binding specificity (15'7). Although these enzymic parameters were obtained under various conditions of buffer species and ionic environ- ment, all factors known to modify the hlich aelis-Menten parameters, they are useful to illustrate the dependence of the enzymic reaction

156. J. J . Maio and H. V. Rickenberg, BBA 37, 101 (1960). 157. J . S . Fruton, Advan. Enzymol. 33, 401 (1970).


on structural features of the substrate. The following substrate requirements have been established:

1. The D-pyranoside ring seems to be essential. Thus, o-nitrophenyl p-D-galactofuranoside is not hydrolyzed (1).

2. Substitution a t C-2, C-3, C-4, and C-6 by groups that are bulkier than the hydroxyl group prevents binding of the galactoside to the active site. Thus, methyl 2-0-methyl P-D-galadoside is neither a substrate nor a competitive inhibitor and the same is true for di- and tri- methylated compounds and for methyl p-D-galacturonide (158). The primary hydroxyl group a t C-6 is easily derivatized and several 6-0- substituted o-nitrophenyl p-D-galactosides were synthesized having -CH,O-PO,H, (159), -CH,O-SO,H (160), -CH,-0-tosyl or -CH,SO,H (161) instead of the hydroxymethyl residue. All were found to be resistant to enzymic hydrolysis. On the other hand, substitution a t C-5 by residues smaller than the hydroxymethyl group is compatible with hydrolyzability. Replacement of this group by hydrogen (158) (as in a-L-arabinosides) by methyl (162) (as in p-D-fucosides) or by methylene (161) (as in the unsaturated 6-deoxy-a-~-arabino hexenopyranoside, cf. Scheme 1) merely reduces the binding specificity of the enzyme. I n contrast, the hydroxyl group a t C-2 seems to be essential for enzymic hydrolysis; 2,4-dinitrophenyl 2-deoxy-2-chloro-~-~-galactoside (163) acts as an inhibitor but is not hydrolyzed enzymically (164). This result cannot be attributed to steric effects, the van der Waals radius of chlorine being smaller by 0.5A than that of hydroxyl, rather i t suggests that this hydroxyl group of the substrate assumes a specific role in the catalytic mechanism.

3. Inversion of the j3-glycosidic linkage to the a confi'guration renders the galactoside resistant to enzymic hydrolysis. However, a-galactoeides are not excluded from the active site as can be inferred from the competitive inhibition of enzymic activity ( 6 4 ) . Epimerization at C-4 to the corresponding glucoside does not completely stop the action of the enzyme, in contrast to earlier reports (158). Dinitrophenyl p-D-glucoside is hydrolyzed, although 10,000 times as slowly as is the corresponding galactoside (164).

158. J. Monod, G. Cohen-Bazire, and M. Cohn, BBA 7, 585 (1951). 159. W. Hengstenberg and M. L. Morse, Carbohydmte Res. 7, 180 (1968). 160. E. Schillinger, Ph.D. Thesis, Freiburg (1965). 161. J. Lehmann and H. Reinshagen, Ann. Chem. 732, 112 (1970). 162. K. Wallenfels, J. Lehmann, and 0. P. Malhotra, Biochem. Z . 333, 209 (1960). 163. W. Hengstenberg and K. Wallenfels, Carbohydrate Res. 11, 85 (1969). 164. W. Hengstenbcrg, P1i.D. Thesis, Freiburg (1966).

20. /3-GALACTOSIDASE 643

4. Replacing the glycosidic oxygen of /3-D-galactosides by sulfur results in a dramatic decrease in the rate of enzymic hydrolysis. Thus, alkyl /3-D-thiogalactosidea and phenyl /3-~-thiogalactoside are not hydrolyzed, although binding of these compounds to the enzyme is not im- paired. Introduction of electron-withdrawing eubstituents in the aromatic ring has a rate-incrcasing effect. This is small with o-nitrophenyl p-D- thiogalactoside (.2), but quite considerable for 1,he 2,4-dinitrophenyl and the 2,4,6-trinitrophenyl derivatives, their rai.cs of hydrolysis being comparable in magnitude with that of the sttmdard substrate o-nitrophenyl /3-D-galactoside (165).

5. I n addition to C-0 and C-S bonds, the enzyme can also catalyze the cleavage of C-F and C-N bonds. Thus, the enzymic hydrolysis of p-D-galactosyl fluoride has been reported, but no kinetic parameters of the reaction have been determined (166). Recently, the structurally related p-D-galactosyl azide was found to be ail excellent substrate with K, 2.83 X

6. The enzyme displays wide tolerance regarlding structural variations of the aglycon moiety which may be another sugar residue, an alkyl or an aryl group. However, the aglycon strongly influences the kinetic parameters of the enzyme. The alkyl 0-p-galactoside series shows a progressive decrease of with increasing chain length, with I<, = 6.9 X 10-3M for methyl, 6.9 X lo-' M for hutyl (64) , and 2 X 1Cr6 M for 2-phenylethyl p- D-galactoside (168), respcctivcly, indicating an increased substrate- enzyme affinity. On the other hand, the decreafe in K, does not parallel an increase in rate of hydrolysis, all three alkyl /3-D-galactosides display- ing a similar V,.

Aryl /3-D-galactosides have I(, values which are in the range of lo-* M (169, 170) with the notable exception of p-nitrophenyl p-D- galactoside. The Michaelis constant of this galactoside was found to be 3 X 10-5M (170), indicating a very high binding specificity of the enzyme toward this substrate, whereas the I<*& 1.2 X lO-'M of 2,4-dinitrophenyl /3-D-galactoside is similar to that for the o-nitrophenyl derivative (1.1 x lo-lM) (164).

44 and kci,t 26 sec-l (167) .

165. K. Wallcnfcls, P. Schacdrl, E. Schillingrr, arid W. Hrnpstmbrrg, Angezu.

166. B. yon Hofsten, BBA 48, 159 (1961). 167. M. L. Sinnot, BJ 125, 217 (1971). 168. K. Wallenfels, A. Schimz, and G. Kurz, unpublished results (1971). 169. 0. M. Viratelle, J. P. Tenu, J . Gamier, and J. Yon, BBRC 37, 1036 (1969). 170. J. P. Tenu, 0. M. Viratelle, J. Gamier, and J. Yon, European J . Biochem.

Chem. Znt. Ed. 3, 450 (1964).

20, 363 (1971).


Although an impressive number of nteta- and para-substituted phenyl P-D-galactosidcs have been synthesized (1'7'1) the effects of substitutents on the rates of hydrolysis have not been thoroughly analyzed. Such data could be important in the elucidation of the mechanism of hydrolysis of these substrates. Treatment of k,,t according to the Hammett equa- tion would yield the reaction constant p and thus provide additional information on the catalytic mechanism of the enzyme. The few data so far available mcrely indicate that thc elcctron-attracting nitro group in meta and para position strongly increases k,.,,, (1'7'0), suggesting a positive p valuc for the enzyme-catalyzed reaction.

Only a few quantitative kinetic data for the enzymic hydrolysis of disaccharides containing D-galactose are available. p-D-Galactosyl (1 + 4) glucose (lactose) and P-D-galactosyl (1 3 6) glucose (allolactose) are excellent substrates with relatively high T', (comparable with that for phenyl P-D-galactoside) albeit surprisingly high I(, (5.5 X M and 8.3 X M, rcspcctivcly) (162). Semiquantitative studies seem to indicate that, in general, the rate of hydrolysis depends on the type of linkage in p-D-galactosyl D-glucoscs and that these rates decrease in thcordcr (1+6)> (1 -4 ) > (1-3) > ( 1 - 1 ) (162) .Sofa r ,wea re aware of only one report in which P-galactosidase from E. wli was found to be inactive toward an O-P-D-galactoside. In this case, desialylated al- acid glycoprotcin, which contains terminal p-D-galactosyl residues linked to 2-acetamido-2-deoxyglucose, was used as a substrate (12).

B. pH DEPENDENCE

Early studies on the influence of pH on the enzymic hydrolysis of o-nitrophenyl P-D-galactoside had given data which were fitted to a bell-shaped curve with maximal enzymic activity between pH 7.2 and 7.4 (64) . A more detailed investigation of the pH effect on V , and K , led to the inference that p-galactosidase has two catalytically important prototropic groups with pK, values in the range of 6.7 and 9.0 (172). However, as was first noted by Reithel and Kim (I%?), the pH dependence of enzymic activity is strongly influenced by divalent ions. These authors could demonstrate that maximal activity for cleavage of o-nitrophenyl P-D-gnlactosidc was shifted from pH 6.8 in the presence of 2 X M Mg2+ to pH 7.4 when no magnesium ions were added. This observation suggests that the mechanism underlying the effect of pH on the kinetic behavior of P-galactosidase is rather complex and that more

171. C. K. De Bluync and J. Wouters-Leywn, Coi-bohyrlrote Res. 18, 124 (1971). 172. I<. Wallcnfels, 0. P. Mnllrotxa, and D. Dabich, Binclwm. Z. 333, 377 (1960).


information is needed to interpret the pH dependence of enzymic hydrolysis.

Recently, the Michaelis-Menten parameters of P-galactosidase-catalyzed reactions have been reinvestigated as a function of pH for the Mgz+ enzyme and for the Mgz+-free enzyme (1%). From the pH profiles of lag kcat, pKm, and log kcat/Km, it was concluded that the activity of both types of enzymes is controlled by a protoriated group which ionizes in the alkaline pH range and by a group which becomes protonated in the acidic pH range. The latter group has a pK < 6 when the enzyme is tested with and without Mg2+ and was tentatively identified as a carboxylate. In contrast, the deprotonation of the second group which dissociates a t higher pH appears to be strongly influenced by magnesium ions. In the Mg2+ enzyme, this group ionizes uith a pK of 8.4, whereas in the absence of Mg2+ the pK was found to be 6.5. This result indicates that the divalent ion obviously increases the affinity of the involved group for the proton. Whether this shift in pK results from a conformational change of the protein or from the interaction of Mg2+ with this particular residue is not known.

C. METAL ACTIVATION

I n his study on P-galactosidase, Lederberg made the important observation that with o-nitrophenyl P-D-galactosicle as a substrate, P-galactosidase is activated by Na+ ions (45) . This finding was confirmed in all subsequent investigations of the effect (64, 65, 133, 172). Moreover, enzyme which was carefully freed from Na+ by exhaustive dialysis against imidazole-HC1 buffer prepared with quartz-distilled water, was found to be virtually inactive (134) . However, activity could be completely restored by addition of Na+ or, to a lesser extent, of K+ and NH,+ ions, indicating that the loss of activity did not result from denaturation of the enzyme.

Neville and Ling analyzed the influence of increasing Na+ concentration on the kinetic parameters of the enzyme (173) . They found that Na+ enhances both the affinity of the enzyme for o-nitrophenyl P-D-galactoside and the maximal rate of hydrolysis. In addition, they could establish that the substrate increases the affinity of the enzyme for Na+ by an amount proportional to the increase in substrate affinity produced by Na+. Thus, there seems to exist a mutual effect of substrate and activator on each other's binding to the enzyme. However, this picture of monovalent cation activation is complicated by the fact that, depending

173. M. C. Neville and G. N. Ling, ABB 118, 596 (1967).


on the substrate, K+ can be a much better activator of enzymic hydrolysis than Na+. When lactose (63), p-nitrophenyl P-D-galactoside (172), or p-nitrophenyl a-L-arabinoside ( 2 ) was used as a substrate, maximal activation was obtained in the presence of K+.

In the case of lactose hydrolysis, Rb+, NH,+, and Cs+ were also found to promote a greater activation (63), whereas the activation of p-nitrophenyl P-D-galactoside promoted by K+ was even inhibited by Na+ (174). Nevertheless, the Michaelis constants for any one of the substrates tested was lowest in the presence of Na+, suggesting that the affinity of the enzyme toward its substrates is greatest with this ion.

Several hypotheses have been advanced to explain these observations. According to Cohen-Bazire and Monod (175) , monovalent ion activation is due to competition of these ions with H+ for some catalytically important prototropic groups which are more active when complexed with the metal ion. Neville and Ling suggested that the activating ion is specifically bound a t a site adjacent to the active site and that this interaction alters the reactivity of the active site and its affinity for the substrate (173). Becker and Evans (174) finally reported that applica- tion of high hydrostatic pressure stimulates the rate of hydrolysis of substrate in the presence of Na+, whereas activation by K+ is considerably reduced under these conditions. They concluded that in the presence of Na+, a decrease in volume accompanies the formation of the enzyme-substrate complex whereas an increase in volume occurs in the presence of K+, and i t was inferred from this finding that the mechanism of enzymic activation by Na+ is different as opposed to the activation by K'. I n summary, no satisfactory picture of the monovalent cation effect can be put forward a t the present time, and i t is conceivable that new data obtained, not from kinetic measurements, but from binding experiments, may provide more meaningful information.

In contrast to the role of monovalent cations, the participation of divalent cations in enzymic hydrolysis has been controversial for a long time. Cohn and Monod reported that Cu2+, Znz+, Ca2+, Mn2+, Co2+, and Sr2+ a t the concentrations tested by them M ) inhibited lactose hydrolysis ( 6 3 ) ; Mg2+ a t a concentration of 2 X 1 e 2 M gave less than 10% stimulation. Rickenberg found that Ca2+, Zn2+, and Ni2+ at low concentrations ( M to lc3 M ) inhibited p-galactosidase activity (176) ; Mg2+ a t concentrations below 10-1 M had no effect OR the enzymic activity but was inhibitory a t higher concentrations. A strongly

174. V. E. Becker and H. J. Evans, BBA 191, 95 (1969). 175. G. Cohen-Bazire and J. Monod, Compt. Rend. 232, 1515 (1951) 176. H. V. Rickenberg, BBA 35, 122 (1959).


activating effect of Mg" was first reported by Hu e t al. (65 ) . Kim and Reithel extended this observation and pointed out that the activation promoted by hlg" is most noticeable at pH <: 7.2 but less pronounced a t higher pH (133). A careful investigation on the effectiveness of divalent cations led to the conclusion that in a system containing Na+ at optimal concentration, all divalent mctals with the exception of the sulfur-complexing heavy metals Cu2+, Pb", and Hg" are strong activators when assayed at low concentrations (134) (Table 111). The least effective metal in this series appears to be Mg", stimulating maximal activity at concentrations between and 10-2M. Since the enzyme is fully activated a t very low concentrations of Zn", Cat'+, or Fez+, and since traces of these ions may be introduced in the assay system by glassware, buffers, substrates, and crude enzyme extracts, it is not surprising that the activating effect of divalent ions went unnoticed.

The results indicate that divalent metals, in contrast to alkali ions, are not necessary for enzymic activity and merely contribute an additional activation. However, there are several observations which suggest that divalent metal ions have an essential role in P-galactosidase- catalyzed reactions. First, the enzyme can be completely inactivated by

TABLE 111 EFFJWT OF I)IVALl:NT k h T - i L IONS ON ~-G-~L~LCTOSID.ISI~: ACTIVITY"

Activi tv

Ion Conc. (MI

x 10-3) mole st bstrat,e mole enzyme x min

Mg2+ 0 10-5 10-4 10-3

10-3 2 x 10-2

Zn2+ 2 x 10-6 2 x 10-4

Fez+ 2 x 10-6 2 x 10-4

MD2+ 2 x 10-6 2 x 10-4

NiP+ 2 x 10-4 Co*+ 2 x 10-4

10-2 Ca2+ 10-6

80 112 138 163 160 150 93 80 155 130 155 155 155 155 135 145

Hydrolysis of o-iiitrophenyl p-D-galactoside (2 X M ) was tested in 0.04 M imidazole HC1 buffer pH 6.8 containing 4 X M NaCl.


treatment with chelating agents (2, ISS), and activity can be restored upon addition of Mg2+ or Ca*+. Second, when p-galactosidase is dissociated in urea solutions containing EDTA, subsequent removal of the denaturing agent leads to the native tetrameric enzyme, which is virtually inactive but is instantaneously activatable by Mg2+ (132). Last, highly purified and seven times recrystallized p-galactosidase was shown to contain about 0.7 g-atoms of Caz+ per mole of subunit of 135,000 molecular weight (2). It cannot, therefore, be excluded that p-galactosidase is a metalloenzyme having a strict requirement for divalent metal ions.

D. KINETICS

Based on their mechanistic model of p-galactosidase catalysis, Wal- lenfels and Malhotra (a) had proposed that the minimal kinetic scheme involves a three-step rather than a two-step mechanism:

Ks ki kaW1 E + S = E S + E S ' - E + P I

+PI It includes rapid binding of the substrate S to yield the Michaelis complex ES, formation of the intermediary complex ES' with concomitant elimination of the aglyconic leaving group PI and hydroiysis of the ES' complex to yield free galactose Pz. Since water is not unique in the sol- volytic step but can be replaced by various alcohols, this kinetic scheme can be expanded to include the transglycosidation reaction

where ES'W and ES" are additional complexes involving the intermediary complex ES' and noncovalently bound water or alcohol, Pz is free galactose, and P, is the galactoside resulting from the transfer reaction. Here, Ks, K,, and K , are the binding constants for the complexes ES, ES'W, and ES'N and kpl k , and k , the first-order rate constants for breakdown of these complexes.

Based on this expanded scheme, Wallenfels e t al. studied the competition of several alcohols with water in the enzyme-catalyzed cleavage of phenyl P-D-galactoside, 2-phenylethyl p-D-galactoside and o-nitrophenyl P-D-galactoside (168). Using the enzyme D-galactose dehydrogenase which permits the simultaneous determination of galactose (Pz) and free aglycon (PI) , they followed the formation of these products from the three donor galactosides in the presence of the acceptor alcohols


methanol, glycerol, 2-mercaptoethanol, P-phmylethanol and tris (hydroxymethyl) aminomethane [ tris] . Depending on the substrate, two types of behavior were observed when the corcentration of alcohol was varied. In the case of phenyl /3-D-galactoside and 2-phenylethyl /3-D- galactoside, increasing the acceptor concentration resulted in enhanced formation of transfer product, although the sum of the rates of hydrolysis and alcoholysis remained constant (Fig. 2) . This result is easy to explain if the formation of the ES’ complex is the rate-determining step of the reaction with k , < k , + k,. Theredwe, the total rate of substrate disappearance does not change when water is replaced by the acceptor, and the increased formation of transfer product can be in- terpreted in terms of simple partition of the intermediate complex ES’ between water and alcohol.

In contrast, when the cleavage of o-nitrophenyl p-D-galactoside was studied, addition of acceptor was found to sl,imulate the enzymic activity of p-galactosidase (Fig. 3 ) . Similar results were reported by Shiffrin and Hunn (177) , although these authors inadvertently com-

Phenol

0.4

I I I 1 I I I , ,- 0.f 0.2

2- Mercaptoethanol ( M )

FIG. 2. Effect of 2-mercaptoethanol on the total reaction rate (liberation of phenol) and hydrolysis rate (liberation of galactose) of phenyl P-D-galactoside cleavage. M/30 sodium phosphate buffer pH 6.8, MgCL lo-’ M , substrate concentration 2 x 10PM.

177. 8. Shifrin and G. Hunn, ABH 130, 530 (1969).


250 -I Methanol

----a /----o-o

-\ Ethanol

n-Propanol

0 5 10 15 2 0 25 3 0 35 40

Concentration (MI

FIG. 3.-Effect of various alcohols on the total reaction rate of o-nitrophenyl p-D- galactoside cleavage in M/30 sodium phosphate buffer pH 6.8, MgCL lo-' 111, substrate concentration 2.5 X 10-'M. Activity in buffer alonc = 100%.

plicated the kinetics of their system by testing the effect of alcohols in tris buffer containing p-mercaptoethanol.

When liberation of galactose and o-nitrophenol was determined in the presence of 2-mercaptoethanol as the acceptor, the data depicted in Fig. 4 were obtained. It is clear that the total cleavage rate of the galactoside increases with acceptor concentration and levels off a t 0;lM 2-mercaptoethanol. At this point, the stimulation of the reaction is about 1.6-fold, whereas the extent of hydrolysis is decreased by 80% compared to the hydrolysis in the absence of acceptor, indicating that most of the rate enhancement is due to the transfer reaction. Analogous results were obtained when the effect of the other alcohols was tested.

These findings suggest that in o-nitrophenyl p-D-galactoside hydrolysis, formation of the intermediary complex ES' and solvolysis of ES' are both rate determining, k 2 and k , being comparablc in magnitude. I n this case, addition of alcohol increases the total rate of solvolysis until it is equal to k,. Recently, similar conclusions were reached by Viratelle e t al. who calculated the kinetic constants for hydrolysis of several galactosides in the presence of methanol. Their data provide convincing evidence that both k , and k:, contribute to the cleavage rate of ortho- and


0.01

0.1 0.2

2-Mercoptoethonol ( M )

FIG. 4. Effect of 2-mercaptoethanol on the total reaction rate (liberation of o-nitrophenol) and hydrolysis rate (liberation of galactose) of o-nitrophcnyl p-D- galactoside cleavage M/30 sodium ~~hosphate huffcr pH 6.8, MgCL M , substrate concentration 2.5 X M .

meta-substituted nitrophcnyl /3-D-galactoside, whereas the reaction rate of several other galactosides tested is solely cetermined by k , (170).

E. MECHANISM OF REACTION

Hydrolyses catalyzed by glycosidases resemble acid-catalyzed hydrolyses in that cleavage of the glycosyl C-1 oxygen bond occurs. This has so far been demonstrated with all glyco3idases investigated. As expected, by carrying out tlic enzymic hydrolysis of o-nitrophenyl p-D- galactoside in H,180 i t was shown that p-galactosidase follows the same scheme (2). A similar conclusion can bc drawii from the enzymic cleavage of di- arid trinitrophenyl P-~-thiogalactosidcs which are excellent substrates of P-galactosidase (165). In this casc, the glycosidic heteroatom is different from the incoming oxygen, and enzyinic hydrolysis yields galactose and the corresponding thiophcnols.

Acid hydrolysis of glycosides is generally believed to proceed via a carbonium ion intermediate. I t is therefore tclmpting to invoke an anal-


ogous process in the catalytic mechanism of glycosidases. Formation of the carbonium ion could be facilitated by two additional factors:

1. Stabilization of the developing transition state carbonium ion as an ion pair by a negative group on the enzyme, and

2. Distortion of the saccharide moiety bound to the active site toward a half-chair which is the most favorable conformation for the cyclic carbonium ion.

So far, experimental data for the involvement of carbonium ion formation in enzymic hydrolysis have only been presented in the case of lysozyme. X-Ray analysis studies have indicated that binding of substrate occurs only if the saccharide ring containing the glycosidic bond to be split is distorted and assumes a half-chair conformation (178, 179, and Chapter 21, this volume). In agreement with this result, i t was concluded on the basis of kinetic (180) and transfer (181) studies that considerable carbonium ion character is involved in the lysozyme-catalyzed hydrolysis of substrates.

That enzymic cleavage of P-galactosides may also proceed via the carbonium ion mechanism was recently suggested by Lee 4182). This author studied the inhibitory action of D-galactal on P-galactosidases from various sources. D-Galactal, unlike P-D-galactosides, asslimes a planar half-chair conformation and thus resembles the postulated carbonium ion intermediate. Since i t can be expected that transition state analogs form a stable complex with the enzyme, and since a strong inhibition of all P-galactosidases investigated was observed, Lee proposed that these enzymes follow a reaction mechanism similar to the one delineated for lysozyme.

Evidence that the postulated protonation of the substrate occurs a t the bridge oxygen and not at the ring oxygen was recently presented by Lehmann and Reinshagen (161). These authors investigated the hydrolysis of the unsaturated derivative o-nitrophenyl 6-deoxy-a-~-A5- arabino-hexenopyranoside (I) and the transfer of the glycosyl residue to glycerol (Scheme 1). Enzymic hydrolysis yields the end product fuconose (11). This result can be explained by two different mechanisms

178. C. C. F. Blake. G. A. Mair, A . C. T. North. D. C. Phillips, and V. R. Sarma,

179. C. C. F. Blake, I,. N. Johnson, G . A . Mair, A . C. T. North, D. C. Phillips,

180. F. U'. Dahlquist. T. Rand-Mcir. and M. A . Raftery. Proc. N o t . Acnd. Sci.

181. J. A . Rupley, V. Gates, and R . Bilbrey, JACS 90, 5633 (1968). 182. Y. C. I m , BBRC 35, 161 (1969).

Proc. Rou. SOC. B167, 365 (1967).

and V. R . Sarma, Pioc. ZZoy. SOC. B167, 378 (1967).

U . 8. 61, 1194 (1968).


I

b H

@

H\ 90 c I

H- C --OH I

HO-C-H I

HO--C-H I c=o I

SCHEME 1

t CHS

-ROH ,O,R t-

'H

OH

@

which involve either protonation a t the glycosidic (A) or the heterocyclic oxygen (B). In the presence of glycerol, however, the transfer product (111) is obtained in excellent yield (48%). Since protonation of the heterocyclic oxygen according to route B leads to an opening of the pyranose ring which is followed by spontaneous tautomerization, and


since recyclization of the intermediate is energetically unlikely, it can be concluded that mechanism B is not involved in the transfer reaction.

Any mechanistic model for the action of /3-galactosidase has to account for the observation that hydrolysis of /3-D-galactosides yields exclusively /I-D-galactose or, with appropriate acceptor, /3-D-galactosides (1, 2, 33) . These findings eliminate a simple displacement mechanism but rcquire a process which involves the retention of conformation a t the anomeric C atom. The following mechanisms can be invoked to cxplain this behavior of the enzyme:

1. Stereospecific solvolysis of a carbonium ion whose orientation a t the active site allows attack by the solvent from only one side.

2. A double displacement mechanism, as suggested by Wallenfels and Malhotra ( 2 ) . It involves protonation of the glycosidic oxygen and attack by a nucleophilic group of the enzyme to form a covalent a-galactosyl-enzyme intermediate. This event is followed by a second displacement reaction with water or an acceptor. Thus, the overall retention of configuration is the result of two inversions. The transition states of these processes are of the fivc-ligand type, thercby avoiding charge sep- aration by a concerted ligand exchange. I t must be emphasized, however, that a t present there is no corroborating evidence for the existence of a glycosyl enzyme in /3-galactosidase-catalyzed reactions.

3. A variation of 2 in which nucleophilic assistance is provided by a functional group of the substrate. This mechanism can be closely sim- ulated by the nonenzymic alkaline fission of aryl glycosides. It has been shown that cleavage of p-nitrophenyl P-D-galactoside in methanolic sodium methoxide occurs via neighboring group participation of the ionized hydroxyl group a t C-2 which yields an intermediate epoxide (185).

Blocking of the C-2 oxygen by a methyl group as in the derivative p-nitrophenyl 2-0-methyl P-D-galactoside causes an enormous decrease in the rate of cleavage. In addition, the character of the reaction is changed. The cleavage of the parent galactoside proceeds with retention of configuration, yielding predominantly 1,6-anhydrogalactose and p- nitrophenol, whereas in the case of the 2-0-methyl derivative free 2-0- methyl galactose and p-nitroanisol are the main reaction products.

Quite similar results were obtained when the base-catalyzed cleavage of 2,4-dinitrophenyl 2-deoxy-2-chloro-/3-~-galactoside was investigated (164). The cleavage rate of the chloro-substituted galactoside is considerably decreased and the reaction clearly involves a nucleophilic attack by the methoxide anion on C-1 of the benzene ring with displace-

183. R. C. Gasman and D. C. Johnson, J. Org. Chem. 31, 1830 (1966)

20. P-GALACTOSIDAGE 655

ment of the galactosyloxy group. The galactoside is bound to the active site of P-galactosidase as indicated by the inhibition of enzymic activity, but it is completely resistant to enzymic hydrolysis. This finding seems to indicate the participation of the 2-hydroxyl group in the catalytic mechanism. It is possible that the developing carboniuin ion is stabilized by the concomitant formation of a C-2 oxyanion which may collapse to a 1,2-epoxide, thus providing thc anchimeric assistance to displace the aglycon. Since neighboring group participation requires a coplanar arrangement of the atomic centers involved, the intermediate epoxide should exist in a half-chair conformation (IV) which is identical with that of D-galactal (V) (see Scheme 2) .

In contrast, the postulated cyclic carboniurn ion (VI) has an overall conformation which is obviously dissimilar t o the conformers (IV) and (V). Thus, the galactal inhibition of P-galactosidasc seems to be in better agreement with the existence of an epoxide transition state. More-

OH

(VI)

SCHEME 2


1 kQ C&OH I OH CHOH H,

+ CHOH I I H CH,OH

SCHEME 3

over, i t is important to note that D-galactal is not only a competitive inhibitor but also a substrate of P-galactosidase. The enzyme was found to catalyze the addition of water to D-galactal to yield %deoxy-P-~-galactose or, in the presence of glycerol, %deoxy-P-~-galactosyl glycerol (184, 186) (Scheme 3) . In addition, isopropyl p-D-thiogalactoside was shown to inhibit the reaction, thus confirming that the addition reaction is a specific P-galactosidase-catalyzed process. These findings extend the catalytic activity of the enzyme to include the addition of water or alcohols to a carbon-carbon double bond. In this regard P-galactosidase bears an interesting similarity with fumarase. This enzyme, which is known to hydrate the double bond of fumarate, was recently shown to catalyze also the hydration of trans-2,3-epoxysuccinate to meso-tartrate (186). Analogous results were obtained with enzymes which catalyze the addition of water to other olefinic acids (187, 188). It is therefore tempting

184. K. Wallenfels and G. Kura, Angeur. Chem. (1972) (in press). 185. J. Lehmann and E. Schroter, Carbohydrate Res. (1972) (in press). 186. F. Albright and G. J. Schroepfer, JBC 246, 1350 (1971). 187. W. G. Niehaus and G. J. Schroepfer, JACS 89, 4227 (1967). 188. W. G. Niehaus, A. Kisic, A. Torkelson, D. J. Bednarczyk, and G. J.

Schroepfer, JBC 245, 3802 (1970).


to speculate that hydration of a C-C double bond and of the corresponding epoxide by the same enzyme is a more general phenomenon and that this special feature may help to clarify the catalytic mechanism of /3-galactosidase.

F. THE ACTIVE SITE

Very little is known about the nature of the catalytic groups of ,8-galactosidase. Based on inhibition experiments with sulfhydryl reagents and on pH activity studies, Wallenfels and Malhotra (2) had proposed that the groups involved in the catalytic mechanism are a sulfhydryl group acting as general acid and an imidazole group providing nucleophilic assistance for the breaking of the glycosidic linkage. However, the pH dependence of maximal velocities does not prove the involvement of specific residues having a corresponding pK in the catalytic site. The apparent PIC could represent the ionization of some group, the dissociation of which is perturbed by other fixed charges in the immediate vicinity or by the environmental polarity of the protein. Moreover, data were recently presented that seem to exclude a direct functional role of SH groups in the catalytic process. Loontiens et d. (101) investigated the effect of the inhibitor o-mercuriphenyl P-wgalactoside which was de- signed as an active-site-directed reagent. It could be established that the compound, although inactivating the enzyme, is completely hydrolyzed and that the reaction product, mercuriphenol, is a more effective inhibitor than the galactoside itself. This proves that the specific binding of the mercurial a t the active site is not followed by the blocking of any catalytically important group. The inactivation by o-mercuriphenyl p-D-galactoside and other mercurials niust be attributed to the reaction of certain SH groups which may be important for maintaining the active conformation of the enzyme.

Destruction of histidine residues by photooxidation with meihylene blue had been studied by Proctor (189). From his findings that enzymic activity was completely lost after destruction of 4 histidine residues, he concluded that imidazole groups play a specific role in the catalytic mechanism. However, the concomitant destruction of other residues such as tryptophan, cysteine, and methionine was nct investigated. Therefore, one is left with the conclusion that neither the chemical modification of the enzyme nor the effect of pH on the enzyme kinetics has provided direct evidence for the nature of the essential residues.

189. M. H. Proctor, BBA 59, 713 (1962).


Recently, i t has been reported that labeling of the enzyme with the active-site-directed reagent N-bromoacetyl p-D-galactopyranosylamine was successful (190). The point of attachment of the label was found to be a methionine sulfur. Although i t remains to be substantiated whether this methionine residue is essential for the catalytical process, these findings provide the first clue to the structure of the active site.

The number of active sites per molecule of enzyme was determined by equilibrium dialysis using the competitive inhibitor 2-phenylethyl p- ~-thiogalactoside and was found to be one per monomer of 130,000 (191).

G . THE TRANSFER REACTION

Substantial evidence indicates that most, if not all, glycosidases can transfer the glycosyl moiety of a substrate to acceptors different from water and that hydrolysis merely represents a special case in which water serves as the acceptor. So far, all p-galactosidases tested were shown to catalyze transgalactosidation reactions (1 , 2,22,27). This raises the question as to whether the enzymes exhibit some sort of specificity toward the acceptor. The p-galactosidase from E . coli, which has been the most thoroughly studied in transfer reactions, has no specific requirements regarding the structure of the acceptor. Transgalactosidation is known to occur with monosaccharides, oligosaccharides, alkyl alcohols, and even phenols (2, 168, 192, 193) whereas no reaction is observed with mercap- tans. However, the rate of transgalactosidation and hence the partition between hydrolysis and transfer is strongly influenced by structural features of the acceptor.

In order to characterize the extent of trnnsgalactosidation, Wallenfels e t al. (168) introduced the transfer number

[free aglycon] [free galactose] [acceptor] Y =

which implies the total reaction rate relat,ive to the rate of hydrolysis in the presence of an acceptor. This ratio was found to depend only on the structure of the acceptor but to be independent of both the structure and the concentration of the donor galactoside. Thus, the data presented in

190. J. Yariv, K. J. Wilson, J. Hildesheim, and S. Blumberg, FEBS Letters 15,

191. M. Cohn, Bacteriol. Rev. 21, 140 (1957). 192. C. Burstein, M. Cohn, A. K6p&s, and J. Monod, BBA 95, 634 (1965). 193. J. B. Pridham and K. Wallenfels, Nature 202, 488 (1964).

24 (1971).


TABLE IV EFFECT OF DONOR CONCENTRATION ON THE TRANSFER NUMBER

Trrtnsfer number

Substrate Methanol Glycerol 2-Mercaptoethanol

o-Nitrophenyl 8-D-galactoside 2.67mM 2 . 2 3!). 1 48 0.26 mM - 41.0 -

Phenyl 8-D-galactoside 12.5 mM 2 . 4 40.0 46 1.25 mM - - 413.6

Table IV show that the transfer number of methanol remains constant whether phenyl P-D-galactoside or o-nitrophenyl P-D-galactoside is used as galactosyl donor, and the same is true for glycerol and 2-mercaptoethanol. Furthermore, i t is evident that the transfer number of glycerol is not changed when different conceiitration,s of the two galactosides are used, indicating that the extent of transfer does not depend on donor concentration.

2-Mercaptoethanol was found to be the mojt effective acceptor of the compounds tested (Table V) , whereas methanol is a rather poor acceptor in transfer reactions. The acceptor activity of tris which is widely used as a buffer ion in P-galactosidase assays is low compared with-that of 2-mercaptoethanol, but i t is high enough to interfere with the determination of kinetic parameters. In general, galactosyl transfer to primary hydroxyl groups is significantly favored. Thus, when hexoses are used as acceptors, the predominant transfer products are the 1 - 6 disaccharides (6). Likewise, transgalactosidation to glycerol yields 1-0- glyceryl P-D-galactoside. Since, in this comlsound, a new asymmetric center is created a t the 2 position of glycerol, the product could be expected to be a mixture of the two stereois:omers I-0-P-D-galactosyl- D-glycerol and 1-0-P-D-galactosyl-L-glycerol. ]However, it could be estab-

TABLE V TRANSFER NUMBERS OF VARIOUE, ALCOHOLS

Acceptor Transfer Number

Methanol Tris 2-Phenylethyl alcohol Glycerol 2-Mercaptoethanol

2 . 2 4 . 5

22.0 40.2 48.0


lished that the main transfer product is the D-glycerol isomer (38) . This result indicates that the mode of binding of glycerol to the enzyme must be highly specific, the alcohol being spatially oriented and positioned a t the binding site in such a way that the transfer reaction can proceed asymmetrically.

H. CONDENSATION REACTIONS

P-Galactosidase readily catalyzes the synthesis of P-D-galactosides by the direct condensation of free galactose and alcohols ( 2 ) . Based on this reaction, a convenient method was worked out which permits the synthesis of various P-D-galactosides labeled with 14C in the glycon moiety (38).

I. INHIBITION STUDIES

Apart from nonhydrolyzable substrate analogs such as most P-D-thio- galactosides, which act by competing for the active site of the enzyme, inhibitors of P-galactosidase may be classified as follows:

1. Heavy metal ions and organomercuric compounds which form stable mercaptide bounds (2 , 101, 189, 194). Their reaction with cysteine residues probably causes structural changes of the protein which are not compatible with enzymic activity.

2. Metal chelators which bind Mg2+ and Ca2+ ions such as citrate ( I & ) , EDTA, and related compounds (2 , 133). In this context, it is noteworthy that o-phenanthroline, which strongly complexes free Zn2+ ions, has no effect on P-galactosidase activity (146).

3. Various alcohols and amines such as 2-mercaptoethanol (121, 195), n-propanol (177) , ethanolamine, ethylenediamine, and mercaptoethylamine (196). In addition to their inactivating effect, these compounds were reported to cause partial or complete dissociation of the tetrameric enzyme to the dimer or the monomer. However, dissociation as well as inactivation were prevented in the case of 2-mercaptoethanol and n-propanol when Mg'+ ions were present. The mechanism of this inhibition is rather obscure. Several of the inhibiting alcohols are even known to be excellent activators of enzymic activity. Since these compounds re-

194. K. Wallenfels, B. Miiller-Hill, D. Dabich, C. Streffer. and R. Weil, Bioehem.

195. S. Shifrin, B. J . Grochowski, and S. W. Luborsky, Nutitre 227, 608 (1970). 196. S. Shifrin, S. W. Luhorsky, and R . J. Grorhowski, RBRC 39, 997 (1970).

Z. 340, 41 (1964).


semble the aglycon moiety of alkyl p-D-galactosides, i t could be conceivable that they inhibit binding of the substrate by competing with the aglycon binding site. However, the dissociation of the enzyme and the antagonistic effect of Mg2+ are difficult to understand. Shifrin et al. suggested that compounds such as mercaptoethylamine may act as chelating agents capable of interacting with metal ions which are essential for stabilization of the tetrameric structure (196). Clearly, more experimental data are desirable for substar;kiating these interesting findings.

J. OTHER p-GALACTOSIDASES OF MICROBIAL, PLANT, AND ANIMAL ORIGIN

In contrast to the enzyme from E . coli, t!ie enzymic properties of p-galactosidase from other microorga,nisms are relatively unexplored. The kinetic parameters, if known a t all, have been mostly determined for o-nitrophenyl P-D-galactoside and lactose. A survey of the data available indicates that most microbial enzymes have pH optima in the range of pH 6.0-7.0 (12, 15, 21, 22, 80, 197). 11; may be noteworthy that for the enzyme from Diplococcus pneumoniae (12) , Saccharomyces lactis ( 2 l ) , and Trichomonas foetus (22) the presence of divalent metal ions is essential for full activity. However, in only one case was a requirement for alkali ions reported (77 ) . The 6-phospho-p-galactosidase from Staphylococcus aureus (80) and the enzymes from Diplococcus pneumoniae (12) , Trichomonus foetus (22) and Neurospora crmsa (197) were found to be inactivated by Hg2+ ions. Trichomonas foetus and Neurospora crassa produce a second p-galactosidase with optimal activity a t pH 4.0 and pH 4.5, respectively (22, 197). The same range of pH optima was described for two p-galactosidases from plants (31, 32) .

During the last few years, a considerable number of reports on animal and especially human P-galactosidases have been published. This stems from the more than academic interest which these enzymes have met and which is due to their physiological importance and to the severe disorders caused by genetically transmitted deficiencies (24, 41 , 198, 199, 200, 201). According to their cellular location two main groups of mammalian P-galactosidases can be distinguished. One group comprises en-

197. G. Lester and A. Byers, BBRC 18, 725 (1965). 198. M. W. Ho and J. S. O’Brien, Science 165, 611 (1969)”. 199. P. A. Ockerman, Clin. Chim. dcta 20: 1 (1968). 200. G. Dawson and A. 0. Stein, Science 170, 556 (1970). 201. J. Rey and J. Frhzal, Arch. F r a n ~ . Pediat. 24, 65 (1967).


zymes which are located in the brush border of the small intestinal mucosa. The second group belongs to the lysosomal enzymes which are ubiquitous in all mammalian cells. The enzyme bound to the brush border is obviously involved in the breakdown of dietary P-galactosides, especially of lactose. It exhibits a rather narrow specificity regarding the aglycone moiety of P-D-galactosides, being most active with lactose as a substrate, although o-nitrophenyl and p-nitrophenyl p-D-galactoside are split as well. This enzyme, which appears to be a true lactase, has been highly purified from the small intestines of calf (86), rat (87), and monkey (88). Since the enzymic properties of the bovine enzyme have been extensively reviewed by Wallenfels and Malhotra ( 2 ) only a brief summary of additional findings will be given here. The human enzyme does not split 6-bromo-2-naphthyl P-D-galactoside. This feature is useful to distinguish the brush border enzyme from a second, cytoplasmic P-galactosidase with similar pH optimum (pH 6) (202) . The lactase from rat is relatively heat labile (87) and is not activated by Na+ (26) .

A special feature of the lactose from human small intestine is its inability to hydrolyze 4- (/3-D-galactosyl) -D-fructose (lactulose) (203) indicating again that this P-galactosidase has some specificity for the aglycon part of its substrate.

/3-Galactosidases localized in lysosomes are characterized, like all lysosomal hydrolases, by their acid pH optimum, which is in the range of pH 3-4. Although most of these enzymes have been studied with the use of low molecular weight synthetic substrates, their physiological role is probably to catalyze the catabolism of complex glycolipids, mucopolysaccharides, and glycoproteins containing p-linked galactosyl residues. This can be inferred from the existence of inborn errors of catabolism, which are characterized by low enzymic activity and by the storage of these products in various organs. Most acid lysosomal P-galactosidases hydrolyze lactose as well as hetero P-D-galactosides such as the phenyl, nitrophenyl, 6-bromo-2-naphthyl and methylumbelliferyl derivatives (55, 85, 90) indicating a wide tolerance for the aglycon. Jungalwala and Robins isolated two P-galactosidases from rabbit brain which were highly active with the nitrophenyl p-D-galactosides and lactose but failed to catalyze the hydrolysis of complex galactosphingolipids (85). On the other hand, Brady et aZ. (40) could demonstrate that a partially purified enzyme from rat intestine which is also present in brain, liver, spleen, and kidney is strictly specific for ceramide trihexosides and does not catalyze the hydrolysis of a number of related sphingolipids or nitro-

202. N. G. Asp, A. Dahlqvist, and 0. Koldovskf, Gastroenterology 58, 591 (1970). 203. A. Dahlqvist and J. D . Gryboski, BBA 110, 635 (1965).


phenyl j3-D-galactosides. These observations suggest that there are specific enzymes for the hydrolysis of galactosphingolipids and that these need not necessarily have galactosidase activity toward synthetic heterosides.

Specific ionic requirements for lysosomal IFgalactosidases have not been investigated. The lysosomal enzyme from rat small intestine was reported to be inhibited by citrate which could suggest a possible role of Ca2+ ions in enzymic hydrolysis. ~-Galactono-1,4-lactone, which was first reported by Conchie et al. (204) to inhibit mammalian P-galactosidase, was found to be a strong competitive inhibitor for lysosomal enzymes from rat intestine (205), ra t epididqmis (204, 2069, ox liver (206), ra t liver (206), rat brain, calf brain (84), and for the neutral brush border lactase from rat intestine (205)

The inhibitory power of the 1,4-lactone is pi-obably due to the formation of small amounts of ~-galactono-1,5-lactc~ne in solution (206, 207) and i t is possible that. this lactone resembles i;he transition state in the enzymic reaction catalyzed by animal P-galactosidases.

ACKNOWLEDGMENTS

We are very grateful to Drs. F. Celada, R. P. Erickson, W. Hengstenberg, W. Messer, K. Paigen, M. B. Rotman, and I. Zabin for having provided manuscripts before publication.

204. J. Conchie, J. Findlay, and G. A. Levvy, BJ 71, 318 (1959). 205. J. Kraml, 0. Koldovsk5, A. HeringovB, V. Jirsov6, K.,KAcl, M. Ledvina, and

206. G. A. Levvy, A. McAllan, and H. J. Hay, BJ 82, 225 (1962). 207. J. Conchie, H. J. Hay, I. Strachan, and G. A. Levvy. BJ 102, 929 (1967).

H. PerlichovB, BJ 114, 621 (1969).

[The Enzymes] Volume 7 || 20 β-Galactosidase

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