Amino acids and protein chemistry 1

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Dr.Ehab Aboueladab

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2. GENERAL STRUCTURE/FORMULA OF AMINO ACIDS1. -carbon bonded to an amino group, acarboxyl group, a hydrogen atom and aside-chain group. - Carbon2. The amino group bonded to the -carbonmakes all AAs for that matter as -aminoCOO- CarboxylHydrogenacids.3. R group determines the identity of thegroup| AA and the role or biologic property of the+H N- C H AA in a protein molecule. 34. Carbon atoms of the side chains are sequentially labeled as , , , , which| AminoR refer to carbons 3, 4, 5, and 6 respectively. Side chain group5. -carbon is a chiral or asymmetric carbon because there are 4 different groups attached to it. 7. At physiologic ph (7.4), the COOH group is6. All AAs except glycine have chiral deprotonated, forming the negatively charged carbons; glycine has 2 H atoms attachedcarboxylate ion ( -CO0-) and the amino group to the -carbon, hence a symmetric is protonated (-NH3+). molecule. 8. With each AA containing at least one amino7. The amino and carboxyl groupsand carboxylic acid group, amino acids are participate in chemical reactions, henceclassified as amphoteric substances and react constitute the functional groups of aminowith both acids and bases. acids and are unique to all amino acids. 3. D & L FORMS (ISOMERS) OF AMINO ACIDS1. Glyceraldehyde is used as a reference compound for D and L isomers of amino acids (similar to carbohydrates).2. In L-glyceraldehyde, the OH- group is on the left side of the molecule; in D-glyceraldehyde, the OH- group is on the right side.3. In L-amino acid as L-alanine, the amino group (NH3+) is on the left side with the carboxyl group at the top of the structure; in D-alanine, the amino group is on the right side with the carboxyl group at the top of the structure.4. In proteins, all of the AAs are in the L-isomer; hence by convention, all AAs are presumed to be in the L configuration unless specifically designated. 4. CLASSIFICATION OF AMINO ACIDS- based on the chemical properties of the side chainsGrp I. AAs with Non-polar or Hydrophobic Side Chains have the tendency to cluster away from H2O (or H2O-hating) Imino group1. Leucine have 4-carbon side chain; the purely ketogenic amino acid.2. Proline a. Its 3-carbon side chain is bonded to the nitrogen of its -amino group and to the -carbon, creating a cyclic or ring structure; this amino acid contains a secondaryrather than a primary amino group, hence an imino acid or a secondary amine. b. This substituted -amino group influences protein folding by forcing a bend in thepolypetide chain. 5. CLASSIFICATION OF AMINO ACIDSGrp I. AAs with Non-polar Side Chains alsothought of as oily or lipid-like.Alanine has a methyl group side chain. 6. CLASSIFICATION OF AMINO ACIDSGrp I. AAs with Non-polar Side Chains1. Methionine side chain contains a sulfur group, similar to cysteine2. Tryptophan side chain contains an indole ring; classified as a neutral amino acid. In Hartnups Disease, there is inability of the intestinal epithelial cells to absorb neutral amino acids like tryptophan excessive amount in the urine impaired synthesis of niacinamide.3. Phenylalanine hydrocarbon group is aromatic; i.e., contains a cyclic group similar to a benzene ring. 7. CLASSIFICATION OF AMINO ACIDSGrp 2. AAs with Neutral Polar Side Chains (Uncharged) participate in hydrogen bonding1. Glycine with very small side chain, hence it causes the least hindrance in a protein (i.e., it does not significantly impinge on the space occupied by other atoms or chemical groups); the simplest AA.2. Serine polar hydroxyl (OH-) group is bonded to aliphatic hydrocarbon groups.3. Asparagine & Glutamine with amide groups in their side chains. 8. CLASSIFICATION OF AMINO ACIDSGrp 2. AAs with Neutral Polar Side Chains (Uncharged)Threonine with 2 asymmetriccarbonsCysteine polar grp. consists of a SH (thiol) grp. which can react with other cysteine SH grps. to form disulfide bridges (-S-S-) or bonds in proteins.Tyrosine hydroxyl grp. is bonded toan aromatic hydrocarbon grp. 9. CLASSIFICATION OF AMINO ACIDSGrp 3. AAs with Carboxyl Groups in their Side Chains (Acidic)The carboxyl grp. (in addition to the one present in all AAs, hence dicarboxylic amino acids) makes these amino acids negatively charged. 10. CLASSIFICATION OF AMINO ACIDS Grp 4. AAs with Basic Side Chains positively charged1. Histidine side chain consists of an imidazole group2. Arginine side chain basic group (guanidino group) is bonded to an aliphatic hydrocarbon tail; the most basic amino acid.3. Lysine side chain amino group is attached to an aliphatic hydro- carbon tail 11. FORMATION OF THE PEPTIDE BOND Slide shows thepeptide bond (C-N)formed between thecarboxyl group ofvaline and the aminogroup of alanine toform a dipeptide, readas valylalanine, notalaninevaline. Again, each componentAA in the dipeptide iscalled a residue ormoiety. 12. PRIMARY STRUCTURE Refers to the linear sequence of aminoacids in a polypeptide chainLEU-GLY-THR-VAL-ARG-ASP-HIS VAL-HIS-ASP-LEU-GLY-ARG-THR1. Although these 2 peptides have the same number and kinds of amino acids, they have different sequence of amino acids and hence have different primary structure.2. The primary structure determines its 3-dimensional structure, properties and functions of a protein.3. This primary structure is determined from the genetic information encoded in DNA. 13. SECONDARY STRUCTURERefers to the hydrogen-bonded arrangement of the backbone of the polypeptide chain.1. There are 2 bonds within an AA with reasonably free rotation: a. bond bet. the -carbon and the aminonitrogen of the residue b. bond bet. the -carbon and thecarboxyl carbon of that residue.2. A peptide chain backbone can be visualizedas a series of playing cards, each cardrepresenting a planar peptide group.3. The cards are linked at opposite corners byswivels, representing the bonds about whichthere is a considerable rotation.4. The angles (phi) and (psi), frequentlycalled Ramachandran angles, are used todesignate rotation around the C-N (-carbonand amino nitrogen) and C-C (-carbon andcarboxyl carbon) bonds respectively.5. The side chains also play vital role indetermining the 3-dimensional shape of theprotein, but only the backbone is consideredin the secondary structure. 14. TERTIARY STRUCTURE OF PROTEINS1. Refers to the shape of the fully folded polypeptide chain, hence distant portions of the secondary structures are close together.2. Exemplified by the structure of MYOGLOBINa. Myoglobin is a compact structure consisting of a single polypeptide chain (153 AAs) and the prosthetic group heme-containing Fe.b. It consists of 8 helices (A to H) stabilized by hydrogen bonds and so are the AA side chains. 15. QUARTERNARY STRUCTURE OF PROTEINS1. Refers to the arrangement of 2 or more polypeptide chains or subunits with respect to one another to form a multisubunit molecule.2. Exemplified by the structure of HEMOGLOBIN a. Hemoglobin is a tetramer consisting of4 polypeptide chains:*2 -chains (blue color) each chainis 141 residues long*2 -chains (green color) each chain is 146 residues longb. The 2 -chains are identical; the 2 -chains are likewise identical.c. Hemoglobin has therefore this overall structure: 22.d. Buried in a crevice within each of the and -chains are the prosthetic groups hemes containing Fe+2. 16. DENATURATION OF PROTEINS1. Refers to the unfolding of the protein hence destruction of its native conformation, esp. of the secondary and tertiary structure.2. This is not accompanied by hydrolysis of the peptide bonds.3. Agents that cause denaturation: a. Heat causes vibrations disruptionof tertiary structure unfolding ordenaturation. b. Extremes of pH decreaseelectrostatic interactions thatmaintain native, active form denaturation. c. Chemical Agents: c1. Chaotropic urea and guanidinium salts c2. Detergents sodium didecyl sulfate (DDS) 17. PROTEIN DIGESTION AND AMINO ACID ABSORPTION 18. OVERVIEW OF DIGESTION OF DIETARY PROTEINS1. Proteins are too large to be absorbed, hence they must be first hydrolyzed to their constituent amino acids. Food2. Digestion occurs via the sequentialStomach action of proteolytic enzymes in the stomach, pancreas & small intestines.Protein HCl PepsinPancreas a. In the stomach, dietary proteins areconverted to polypeptides & somePolypeptidesfree amino acids by pepsin. HCO3- b. In the small intestines, trypsin,Trypsinchymotrypsin, elastase & carboxy- Chymotrypsinpeptidases A & B produce oligopep-Elastasetides in the presence of HCO3- (which CarboxypeptidasesSmallneutralizes the stomach acid &A&Bintestineraises the pH for activation of the Oligopeptideszymogens). c. Final digestion occurs via amino-Aminopeptidasespeptidases to produce free AAs and Bloodsmaller peptides like di- and Di- and tri- Di- and tri-tripeptides. peptidases Amino peptides acidsAmino3. Amino acids are then absorbed by the intestinal epithelial cells, brought to + acids the liver (via the portal system) forAmino metabolism or release into theacids general circulation. Intestinal epithelial cell 19. ACTIVATION OF THE GASTRIC AND PANCREATIC ZYMOGENS ProenzymesActive Enzymes H+ (parietal cells) PepsinogenPepsin Enteropeptidase Trypsinogen (enterokinase)Trypsin trypsin ChymotrypsinogenChymotrypsin ProelastasetrypsinElastase Procarboxypeptidase A & B trypsin Carboxypeptidae A & B1. The gastric and pancreatic zymogens involved in protein digestion are secreted in their inactive precursors or zymogen forms initially and then converted to their active forms.2. Pepsinogen, secreted by the chief cells of the stomach, is converted to pepsin via HCl (from gastric parietal cells) and autocatalytically by pepsin.3. Trypsinogen is converted to the active form trypsin by enteropeptidase (formerly enterokinase) from intestinal mucosal cells.4. Thereafter, trypsin cleaves the other pancreatic zymogens, producing their active forms.5. Trypsin is therefore the master or common activator of the pancreatic zymogens. 20. + NH3N - terminus ACTION OF THE DIGESTIVEamino-|HCRpeptidases| PROTEASES C=O| NH|PheA. Endopeptidases hydrolyze peptide bonds pepsin H C R Tyr| Glu at various points within the polypeptide chain. C=O| Asp 1. Pepsin preferentially cleaves peptide bondsNHin which the carboxyl (carbonyl) group is |ArgtrypsinHCRprovided (or contributed) by aromatic AAs | Lys(Phe, Tyr) and acidic AAs (Glu, Asp).C=O| NH| Phe2. Trypsin specifically cleaves peptide bondschymotrypsin H C RTyr in which the carboxyl group is provided by |GluC=O positively charged AAs (Arg, Lys). |Leu NH|Ala3. Chymotrypsin favors hydrolysis of peptideelastase H C R Gly| bonds in which the carboxyl group is providedC=OSer by aromatic AAs (Phe,Tyr) and hydrophobic| NH AAs (Glu, Leu).|HCR|4. Elastase cleaves elastin (for which it wasC=O carboxy-|peptidase A named) and peptide bonds whose carboxyl NH(hydrophobic) group is provided by AAs with small sidecarboxy- | carboxy-peptidases H C R chains (Ala, Gly, Ser).|peptidase B COO-C - terminus (Arg Lys) 21. + NH3N - terminusACTION OF THE DIGESTIVE amino-|HCRpeptidases|PROTEASESC=O| NH|PheB. Exopeptidases hydrolyze peptide bondspepsin H C R Tyr| Glu at outer ends of a polypeptide chain. C=O| Asp 1. Carboxypeptidase A hydrolyzesNH|peptide bonds at the C-terminus oftrypsinHCRArg| Lyshydrophobic AAs (Ala, Ile, Leu, Val).C=O| NH Phe2. Carboxypeptidase B hydrolyze peptide | chymotrypsin H C RTyr bonds at the C-terminus of Arg and Lys.|GluC=O|Leu NH3. Aminopeptidases, coming from the |Ala brush border of the epithelial mucosa, elastase H C R Gly| complete protein digestion byC=OSer| hydrolyzing one amino acid at a timeNH| from the N-terminus of peptides to HCR| produce free amino acids, di- and C=O carboxy- tripeptides. |peptidase A NH(hydrophobic) carboxy- |peptidases H C R carboxy-|peptidase B COO- (Arg Lys)C - terminus 22. TRANSEPITHELIAL AMINO ACID TRANSPORT Intestinal Amino lumen acid Na+1. It is an active process, similar to CHO digestion in the intestinal epithelial cells.Brushborder2. In the brush border membrane is a semi-specific Na+-dependent transport Amino Na+ protein that carries or pulls with it the AA acid as it moves along concentration gradient (since Na+ conc. in the lumen is > than itsActive concentration in the inside of the cell).transporterATP Na+3. Na+ isthen pumped into the serosal side by the Na+-K+-ATPase system which is ADPK+ counterbalance by K+ moving in the+Pi K+ opposite direction.4. The accumulated AA inside the cell is then SerosalsideFacilitated transported down its concentration transporter gradient via facilitated transporters intoAmino the serosal side brought to the liveracid Portal via the portal vein for metabolism or forvein distribution to other tissues. 23. TRANSEPITHELIAL AMINO ACID TRANSPORT Intestinal Amino lumen acid Na+Brush5. There are different Na+-dependent AA border transport proteins in the apical brush border membrane of the intestinal Amino Na+ epithelial cells, each transport systemacid transporting a group of closely related amino acids: Active a. Transport system for neutral AAs. transporter b. Transport system for proline andATP Na+ hydroxyproline. c. Transport system for acidic AAs.ADPK+ d. Transport system for basic AAs. +Pi K+ (Lys, Arg, Ornithine) and cystine.6. Some amino acids use facilitatedSerosal transport carriers.Facilitatedside7. Most amino acids are transported by moretransporter than one transport system.Amino9. Ingested dietary amino acids by man areacid Portal used primarily for synthesis of proteins.vein 24. BIOSYNTHESISOF THENUTRITIONALLYNON-ESSENTIAL AMINO ACIDS 25. CLASSIFICATION OF AMINO ACIDSGlucogenicGlucogenicand Ketogenic (13 AAs) Ketogenic(2 AAs)(5 AAs) Alanine AsparagineNon-essential Aspartate Cysteine PVT TIM HALL GlutamateTyrosine Glutamine Glycine Proline SerineEssential ArginineIsoleucine HistidinePhenylalanineLeucine MethionineTyrptophan Lysine Valine Threonine 26. OVERVIEW OF THE BIOSYNTHESIS OF THE NON-ESSENTIAL AMINO ACIDSGLUCOSEGlycine Glutamate Methionine3-Phosphoglycerate Serine Cysteine AsparaginePyruvateAlanine Acetyl CoAGlutamine PhenylalanineTyrosine CitrateAspartateOxaloacetateGlutamineIsocitrate Proline-ketoglutarateGlutamateArginine1. There are 11 non-essential amino acids of the 21common amino acids.2. Non-essential because they can be synthesized in sufficient amounts by the body from: a. Amphibolic intermediates of:a1. Glycolysis i. 3-phosphoglycerate - Cysteine, Serine, and Glycine ii. Pyruvate - Alaninea2. Citric Acid Cycle i. -ketoglutarate Glutamine, Glutamate, Proline and Arginine ii. Oxaloacetate Aspartate and Asparagine b. Phenylalanine Tyrosine 27. I. AMINO ACIDS DERIVED FROM GLYCOLYSISC1OO- A. Serine IC1OO-H C2 OH I I 3-phosphoglycerate C2 = OC3H2 dehydrogenaseI IC3H2GlycolysisIO - PO32-Glucose3-Phosphoglycerate O - PO32- 1. 3-phosphoglycerate (from3-Phosphohydroxypyruvateglycolysis) is converted to 3-phos- NAD+ NADH COO-(keto acid)phohydroxypyruvate, a keto acid, via + H+ | H3N+ - C H3-phosphoglycerate dehydrogenase|with NAD+ as a hydrogen acceptor Glycine CH2reduced to NADH2. |PLP D- serineCH2 2. Followed by transamination with (CNS neurotransmitters) |glutamate as the amino group donorSelenocysteineCOO-to 3-phosphohydroxypyruvate, Glutamateforming 3-phosphoserine and - aminotransferaseketoglutarate in the presence of -KetoglutaratePLP,a cofactor, via anaminotransferase. 3. Finally, phosphoserine phosphatase C1OO- C1OO-Phosphoserinehydrolyzes the phosphate group+ I + Iphosphatase(dephosphorylation) to form serine. H3N C2 HH3N C2 H 4. Serine is a precursor of the neuro-IItransmitters glycine and D-serine; C3H2C3H2also a component of the unusual AAI Iselenocysteine found in glutathioneOH Pi O - PO32-peroxidase. Serine3-Phosphoserine 28. I. AMINO ACIDS DERIVED FROM GLYCOLYSIS Other Pathway For Serine H20 Serine hydroxymethylCH2OHH2C NH3+transferase ||H C NH3+COO-PLP | GlycineCOO- H20SerineN5, N10 CH2 TH4(N5-N10-Methylene THF) TH4(Tetrahydrofolate) 1. Serine may also be formed reversibly from glycine by transfer of ahydroxymethyl group via serine hydroxymethyl transferase in thepresence of PLP and tetrahydrofolate (TH4), forming N5, N10-MethyleneTH4. 2. The demand for serine and glycine and the amount of N5, N10-MethyleneTH4 determine the direction of the reaction. 29. I. AMINO ACIDS DERIVED ROM GLYCOLYSIS B. Glycine the Major PathwayCOO-PLP Serine hydroxymethylCOO-Itransferase +I3-Phospho- +H3N C H H3N C H glycerateI ICH2OH H SerineGlycineHHNN C8 7 CH2 CCH2 6HH C5 9C 5 Reactive part of N CH2 N CH2H I I I tetrahydrofolate10 N Methylene 10H2C NTetrahydrofolateH(FH4) N5-N10-Methylenetetrahydrofolate(N5-N10-CH2-H4 folate) 1. Serine, coming from the glycolytic intermediate 3-phosphoglyerate, is converted reversibly toglycine via serine hydroxymethyltransferase, with PLP as a coenzyme and tetrahydrofolate (FH4). 2. The reaction involves the transfer of a one-carbon unit from serine with tetrahydrofolate as anacceptor. 3. FH4 is converted to N5-N10-methylenetetrahydrofolate (methylene, a one-carbon unit is bound to 2 ofthe nitrogens of the carrier molecule). 4. Hence FH4 is a carrier of one-carbon units (-CH2) in metabolic pathways; the other carriers arebiotin, a carrier of CO2 in carboxylation reactions and S-adenosylmethionine (SAM), a carrier ofmethyl groups (-CH3) in methylation reactions. 30. I. AMINO ACIDS DERIVED FROM GLYCOLYSISMinor PathwayB. GlycineCH3 H| |OHCCC PLPH2C NH3+| ||O-OH NH4+COO- Threonine Glycine Threonine is degraded into glycine in the presence of PLP s a cofactor. 31. COO-+ II. AMINO ACIDS DERIVED FROM GLYCOLYSISH3N C HC. Cysteine ICH2 OH -OOC CH CH SCH2SH |2| | | IH C NH3+ +NH CH2 3 CH2 CH2|| | ICOO- CH2 CH2 H3C - SSerineMethionine| |+ H C NH3 + H C NH3ATP| | MethionineCOO- COO-adenosyltransferaseCystathioneCystathionineHomocysteine PLP -synthaseH2O H2O PLP S-adenosyl methionine def-Cystathionase (SAM; Adomet)adenosineCystathionuria NH4+ Methylase CH3 Adenosylhomo- S-adenosyl-Ketobutyratecysteinasehomocysteine -OOC CH CH SH(SAH)2 + |1. Methionine (an essential AA) condenses with ATP via NH3 methionine adenosyltransferase to form S-adenosylmethio-Methio- nine (SAM; Adomet); methyl group of SAM is cleaved bySerinenine methyltransferase to form S-adenosylhomocysteine (SAH); Adenosine group of SAH is cleaved by adenosylhomocys Cysteine teinase to form homocysteine.2. Serine condenses with homocysteine (from methionine) to form cystathionine via cystathione -synthase with PLP as a co-factor. 32. COO-+ II. AMINO ACIDS DERIVED FROM GLYCOLYSISH3N C HC. Cysteine ICH2 OH-OOC CH CH SCH2SH | 2|| | IH C NH3++NH CH23 CH2 CH2||| ICOO- CH2CH2 H3C - SSerineMethionine||+ H C NH3 +H C NH3ATP|| Methionine COO- COO-adenosyltransferaseCystathione CystathionineHomocysteine PLP -synthase H2OH2O PLP S-adenosyl methioninedef-Cystathionase (SAM; Adomet)adenosine Cystathionuria NH4+ Methylase CH3 Adenosylhomo- S-adenosyl-Ketobutyratecysteinasehomocysteine-OOC CH CH SH(SAH) 2+ |3. Cystathione is then cleaved by PLP-dependent -cystathio-NH3 nase into -ketobutyrate (and NH4+) and cysteine. Methio-4. Serine contributes the carbons and nitrogens for cysteine;Serinenine methionine provides the sulfur of cysteine via transulfuration - one of the routes used for methionine catabolism. Cysteine5. Because methionine is an essential amino acid, cysteine synthesis can be sustained only if the dietary intake of methionine is adequate. 33. COO-+ II. AMINO ACIDS DERIVED FROM GLYCOLYSISH3N C H C. Cysteine ICH2 OH -OOC CH CH SCH2 SH|2 || | IH C NH3+ +NHCH23 CH2 CH2| || ICOO-CH2CH2 H3C - S SerineMethionine ||+ H C NH3+H C NH3ATP || MethionineCOO-COO-adenosyltransferaseCystathioneCystathionineHomocysteine PLP -synthaseH2O H2O PLP S-adenosyl methionine adenosine def-Cystathionase (SAM; Adomet)Adenosylhomo-Cystathionuria NH4+ Methylase CH3 cysteinase -Ketobutyrate S-adenosylhomocysteine -OOC CH CH SH(SAH)2 + | NH36. A genetic deficiency of cystathione -synthase orMethio-SerineninePLP causes cystathionuria or the presence of cystathione in the urine; a benign disorder with noCysteine clinical abnormalities. 34. II. AMINO ACIDS RELATED TO TCA INTERMEDIATESA. Amino Acids Related to -ketoglutarateCOO- COO- |1. Glutamate| H3N+ - C H C=O NAD(P)+ NAD(P)H| | NH4+ CH2 CH2| |CH2 CH2| | GlutamateCOO- COO-dehydrogenaseGlutamate-ketoglutarate Synthesis of other AAs Glutamine, Proline Arginine, OrnithineGlutathione1. Reductive amination of -ketoglutarate to glutamate via glutamate dehydrogenase is a freely reversible reaction; NAD+ or NADP is a cofactor.2. The 5-carbons of glutamate came from -ketoglutarate which also came from glucose.3. Glutamate is used for the synthesis of other amino acids: glutamine, proline, arginine, ornithine and glutathione (an antioxidant). 35. COO- - carboxyl group |GLUTAMATE AS ASHH3N+ - C HI| PRECURSOR OF CH2 CH2 |GLUTATHIONE I +CH2 HC NH3| - carboxyl groupI COO-COO- GlutamateCysteine-Glutamylcysteine-glutamylcysteineATPCOO-1. Glutamate, in the presence of synthaseI Glutathione H3N C H ATP, condenses with cysteine synthase IATP to form the dipeptide -gluta-ADP + Pi HADP + Pi mylcysteine. GlycineSulfhydryl group SH2. Followed by the addition of of cysteine I O CH2 O glycine, again in the presenceII H III H -carbonC N CH C N CH2 of ATP to form the tripeptide - or I I glutamylcysteinylglycine; the C3H2 COO- letter refers to the 3rd carbon3rd carbonI in the molecule, counting the C2H2I + one bonded to the amino groupHC1 NH3 as the 1st carbon (recall that I is the 3rd letter in the GreekCOO- alphabet).Glutathione (-Glutamylcysteinylglycine) 36. GLUTATHIONE AS AN ANTIOXIDANTH2O2 2 - Glu Cys - Gly - Glu Cys -Gly IISulfhydryl Glutathione SHperoxidase2 H2OS DisulfidegroupGlutathione I bond (GSH; reduced form)SI1. The sulfhydryl group of glutathione - Glu Cys - Gly (GSH; reduced form) reduces or scavenges H2O2 (an oxidizing agent)Glutathione disulfide to H2O, catalyzed by glutathione(GSSG; oxidized form) peroxidase, forming glutathione NADPH disulfide (GSSH; oxidized form)+ H+ Glutathionereductase consisting of 2 molecules of the reduced form joined together by aNADP+ disulfide bond between the SH2 - Glu Cys - Gly groups of the 2 cysteine residues.I SH2. Reduced glutathione (GSH) is 2 GSH regenerated via glutathione (reduced form) reductase at the expense of NADPH. 37. GLUTATHIONE IS INVOLVED IN THE TRANSPORT OF AMINOACIDS ACROSS PLASMA MEMBRANES Amino acid-GlutamyltranspeptidaseOutside -Glu-Cys-Gly Cys-Gly -Glu-Amino acidPlasma membraneInside-Glu-Cys-Gly-Glu-Amino acid Cys-Gly -GlutamylAmino (glutathione)H2O cyclotransferase acid Cysteine 5-Oxoproline ADP + Pi5-oxopro- ATP + H2O+ 2 H+ATP + Glycine linase ADP + Pi+ 2 H+-Glu-Cys-Gly -Glutamyl- cysteine L-Glutamate (glutathione) Glutathione -Glutamylcysteinesynthasesynthase 1. Transport of amino acids uses the -glutamyl cycle (Meister Cycle). 2. Membrane-bound -glutamyltranspeptidase catalyzes the transpeptidation of -glutamyl residue from glutathione to the amino acid, forming -glutamyl-amino acidand cysteinylglycine, the remaining portion of glutathione. 3. The -glutamyl-amino acid (a dipeptide, consisting of 2 amino acids glutamate and theamino acid to be transported) is transported into the cytoplasm to form 5-oxoproline. 4. Once inside the cell, the cytoplasmic enzyme -glutamylcyclotransferase releases theamino acid and cyclizes the -glutamyl group to form 5-oxoproline, which is convertedto glutamate via 5-oxoprolinase in the presence of ATP. 38. GLUTATHIONE IS INVOLVED IN THE TRANSPORT OF AMINOACIDS ACROSS PLASMA MEMBRANES Amino acid-GlutamyltranspeptidaseOutside -Glu-Cys-GlyCys-Gly-Glu-Amino acidPlasma membraneInside-Glu-Cys-Gly-Glu-Amino acidCys-Gly-GlutamylAmino (glutathione)H2O cyclotransferase acidCysteine5-Oxoproline ADP + Pi5-oxopro- ATP + H2O+ 2 H+ATP+Glycinelinase ADP + Pi+ 2 H+-Glu-Cys-Gly -Glutamyl- cysteine L-Glutamate (glutathione) Glutathione -Glutamylcysteinesynthasesynthase 5. To complete the -glutamyl cycle, cysteine condenses with glutamate via -glutamylcysteine synthase, again in the presence of ATP, forming -glutamylcysteine. 6. -glutamylcysteine then condenses with glycine via glutathione synthaseagain in the presence of ATP, forming and resynthesizing glutathione. 39. II. AMINO ACIDS RELATED TO TCA INTERMEDIATESA. Amino Acids Related to -ketoglutarate2. Glutamine COO-ATPNH2|ADP + Pi|H3N+ - C H NH + C=O 4| |-ketoglutarate CH2Glutamine synthaseCH2| |CH2 Glutaminase H C NH3+| | COO-COO-Glutamate NH4+H2O Glutamine1. Glutamine coming from transamination of -ketoglutarate, is aminated to glutamnine via glutamine synthase by adding free NH4+ (as the amino group donor) to the carboxyl group (-COOH) of the side chain of glutamate in the presence of ATP.2. In the presence of glutaminase, glutamine is deaminated to glutamate.3. Asparagine is the other amino acid that is synthesized from amination of aspartate. 40. II. AMINO ACIDS RELATED TO TCA INTERMEDIATESA. Amino Acids Related to -ketoglutarate + NH3. Proline-COO CH2 3CH2 CH COO- ATP Glutamate NADH + ADP + PiH+1. Glutamate is phosphory- NADH lated by ATP and then + H+ NAD+ + converted to glutamateNH NAD+ H - C CH2 CH2 3 CH COO- semialdehyde via the II Glutamate semialdehyde reduction of side chain O carboxyl group (-COOH) Spontaneous cyclization to an aldehyde. H2CCH2 HC +CH COO-2. Followed byN spontaneous H cyclization of to 1- NADPH 1-Pyrroline 5-caboxylate + pyrroline 5-carboxylate.FAD . 2H Pyrroline-5-H+ carboxylate3. This cyclic compound isreductase FADH2CCH2 then reduced by NADPH NADP+ H2C+CH COO- to proline via pyroline-5- N carboxylate reductase. H2Proline 41. II. AMINO ACIDS RELATED TO TCA INTERMEDIATES A. Amino Acids Related to -ketoglutarate1. Glutamate, coming from 4. Arginine -ketoglutarate, is+ NH3 -Ketoglutarate converted to glutamate | semialdehyde.H C CH2 CH2 CH COO-||2. Followed by transamina-O Glutamate semialdehydeGlutamate tion into ornithine (an intermediate of the ureaOrnithine cycle) arginine. Transanimationaminotransferase +3. The amounts of arginineNH3 generated by the urea | cycle is adequate only H3N+ - CH CH CH CH COO- 222 for the adult and are Ornithine insufficient for growth, hence during periods of Urea+ rapid growth as inNHcycleNH3 infancy and childhood,||| arginine becomes an H2N C CH2 CH2 CH2 CH COO- essential amino acid.Arginine 42. II. AMINO ACIDS RELATED TO TCA INTERMEDIATESB. Amino Acids Related to Oxaloacetate Aspartate & Asparagine1. Oxaloacetate is transami- COO- nated to aspartate with PLPCOO-| | as a cofactor in a freely CH2 reversible reaction. CH2|Transanimation|H C NH3+C=O2. Aspartate is then aminated to| asparagine via asparaginePLP | COO- COO- synthetase in the presence ofATPGlutamineAspartate NH4+Oxaloacetate ATP and glutamine as a source of nitrogen (or the Asparagine Asparaginase amino group donor).synthetase3. Leukemic cells require O H2OGlutamate || asparagine for their growth, hence asparaginase has been AMP + PPiC NH2 used as an anti-tumor agent.| CH2It acts by converting|asparagine to aspartate in the H C NH3+blood, thereby decreasing the|amount of asparagineCOO-available for tumor growth. Asparagine 43. III. TYROSINE COMING FROM PHENYLALANINE GTPBiosynthesis+ H HNH3 N NH I NAD+H2N1 - CH2CHCOO- 2 8 7 HN 3 4 6 H 9 10 Phenylalanine5CH CH CH3NIIHI I O2O OH OHDihydropteridine Phenylalanine Tetrahydrobiopterin reductasehydroxylase(BH4) H H2O H2NNN H HNADH+ + H+ H NH3 NI NCH CH CH3 HO -- CH2CHCOO- 3 II 5 II OOH OHQuinonoid dihydrobiopterin (BH2)Tyrosine1. Phenylalanine is reduced to tyrosine via phenylalanine hydroxylase (PAH), a mixed- enzyme oxidase with 2 activities a. Activity I: Reduction of O2 to H2O and of phenylalanine to tyrosine (molecular O2donates one atom to H2O and one atom to the product tyrosine). 44. III. TYROSINE COMING FROM PHENYLALANINE GTPBiosynthesis + H H NH3 N NHI NAD+H2N1- CH2CHCOO- 2 8 7 HN 3 4 6 H 9 10Phenylalanine5CH CH CH3NIIHI IO2O OH OHDihydropteridinePhenylalanine Tetrahydrobiopterin reductase hydroxylase(BH4) HH2OH2N NN H HNADH + + H+ HNH3 N I NCH CH CH3 HO - - CH2CHCOO- 3 II 5 II OOH OHTyrosine Quinonoid dihydrobiopterin (BH2) b. Activity II: Reduction of the co-factor tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2); the Hatoms on carbons 3 and 5 of BH4 are absent in BH2.2. BH4 is synthesized in the body from GTP; BH4 is usually used in the hydroxylation of aromatic amino acids like phenylalanine, tyrosine and tryptophan (with aromatic rings).3. BH2 is reduced back to BH4 via an NADH-dependent dihydrobiopterin reductase for phenylalanine to continue forming tyrosine. 45. III. TYROSINE COMING FROM PHENYLALANINEGTPBiosynthesis +H HNH3N NH INAD+H2N1 - CH2CHCOO-2 8 7HN 3 4 6 H 9 10 Phenylalanine 5CH CH CH3 N IIHI I O2 O OH OH DihydropteridinePhenylalanineTetrahydrobiopterinreductase hydroxylase (BH4)H H2O H2N NN H H NADH++ H+ H NH3NINCH CH CH3 HO -- CH2CHCOO-3 II 5 IIOOH OH TyrosineQuinonoid dihydrobiopterin (BH2)4. Tyrosine is unique because unlike all the other non-essential AAs which are synthesized from certain intermediates of glycolysis (3-phospholycerate and pyruvate) and Krebs Cycle, it results from a simple one-step hydroxylation of phenylalanine.5. Hence the presence of dietary tyrosine decreases the need for phenylalanine. 46. IV. BIOSYNTHESIS OF HYDROXYPROLINE AND HYDROXYLYSINECOO-I COO-CH2II CH2CH2 II CH2C=O IICOO-COO- -ketoglutarate [18O] Succinate 18OFe2+18OH 2 Pro (Lys) Ascorbate Pro (Lys)Prolyl hydroxylase(Lysyl hydroxylase)1. Peptide-bound proline and lysine are hydroxylated by prolyl hydroxylase and lysyl hydroxylase respectively (found in skin and skeletal muscles, including granulating wounds), in the presence of molecular O2 (one atom of O2 is incorporated into proline or lysine; the other atom into succinate), ascorbate , Fe+2 and -ketoglutarate.2. Hydroxyproline and hydroxylysine are present principally in collagen; collagen is a glycoprotein in all tissues and organs provides the framework that gives tissues their form and structure.3. A deficiency of Vitamin C or hydroxylase results to scurvy.