Transcriptional coactivator protein p300: Kinetic characterization of ...

Transcriptional coactivator protein p300: Kinetic characterization of its histone acetyltransferase activity✝

Paul R. Thompson*, Hisanori Kurookaδ, Yoshihiro Nakataniδ, Philip A. Cole*

*Department of Pharmacology and Molecular Sciences, Johns Hopkins University, 725 N.

Wolfe St., Baltimore, MD, USA 21205; δDana-Farber Cancer Institute, Boston, MA 02115

Running Title: Kinetic characterization of p300

✝ This work was supported in part by the Canadian Institutes of Health Research (P.R.T.) and NIH (P.A.C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Address correspondence to P.A. Cole, email [email protected]; tel (410) 614-0540

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 9, 2001 as Manuscript M104736200 by guest on A

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ABSTRACT The p300/CBP family members include human p300 and CREB binding protein,

which are both important transcriptional coactivators and histone acetyltransferases.

While the role of these enzymes in transcriptional regulation has been extensively

documented, the molecular mechanisms of p300 and CBP HAT catalysis are poorly

understood. Herein, we describe the first detailed kinetic characterization of p300 using

full-length purified recombinant enzyme. These studies have employed peptide

substrates to systematically examine the substrate specificity requirements and the kinetic

mechanism of this enzyme. The importance of nearby positively charged residues in

lysine targeting was demonstrated. The strict structural requirement of the lysine side

chain was shown. The catalytic mechanism of p300 was shown to follow a ping-pong

kinetic pathway and viscosity experiments revealed that product release and/or a

conformational change were likely rate-limiting in catalysis. Detailed analysis of the

p300 selective inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.

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INTRODUCTION

Since the initial revelation that yeast Gcn5p1 (Gcn = global control non-repressed)

is a histone acetyltransferase (HAT) (1), and the subsequent determination that a number

of other transcriptional co-activators possess HAT activity, there has been an explosion of

interest into the role of protein acetylation in vivo (reviewed in 2-5). More than 20 HATs

have now been identified and these have been classified into one of 5 families (GNAT,

MYST, TAF,,250, nuclear receptor coactivators, and p300/CBP), primarily based upon

amino acid sequence homology (2).

The p300/CBP family of HATs is represented by two proteins, p300 and CREB

binding protein (CBP), that share considerable sequence homology over their entire

length; human p300 and CBP proteins share 91% sequence identity (6). The coactivator

p300 is a 2414 amino acid protein that was initially identified as a nuclear binding target

of the E1A oncoprotein (7), whereas CBP is 2441 amino acids long and was originally

identified as a transcriptional coactivator that bound to phosphorylated CREB in mice (8).

Subsequently Arany et al (6) noted the significant sequence homology between these two

proteins and suggested that these two large proteins (~265 kDa) might be functionally

equivalent, which has since been confirmed in many cases (9). The protein pair,

p300/CBP, as it is now commonly referred to, is important for both cell cycle progression

and cellular differentiation, and possesses a number of distinct functional domains that

have been shown to interact with components of the RNA pol II holoenzyme,

1 The abbreviations used are: Gcn, global control nonrepressed; Cit, citrulline; Orn, ornithine; Hnl, 6-hydroxynorleucine; Iso, N-ε-isopropyl-lysine; Cap, 1-aminopropanol-cysteine; D-Lys, D-lysine; CREB, cyclic adenosine monophosphate response element binding protein.


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transcription factors, and nuclear hormone receptors and their co-activators (2, 10-19)

(Figure 1).

Following the determination that PCAF (p300/CBP associated factor) is a bona

fide HAT (20), Ogryzko et al (21) and Bannister and Kouzarides (22) independently

demonstrated the intrinsic HAT activity of p300 and CBP (Figure 2). For p300, the

domain responsible for this activity has been localized to a region encompassing amino

acids 1195-1673 (21). Of the known HAT families the GNAT family is the best

characterized mechanistically and structurally (2,23). At the mechanistic level, it has

recently been shown that two distantly related GNAT family members, P/CAF and

serotonin N-acetyltransferase, as well as GCN-5 itself employ ternary complex

mechanisms that involve the ordered binding and release of substrates and products (24-

27).

These results suggest that the mechanistic paradigm for this family of enzymes is

the direct transfer of the acetyl group from acetyl-CoA to the substrate lysine nucleophile.

Recently, the crystal structure of the MYST (MOZ, YBF2/Sas3, Sas2, Tip60) family

member Esa1 (Essential Sas2-related acetyltransferase) in complex with CoASH has been

determined and the structure revealed that this protein and GNAT family members adopt

the same overall fold (28). The structural homology observed between these two

families, despite limited primary structure homology, has led to the suggestion that other

HAT families, including p300/CBP, will have the same protein fold and employ a similar

catalytic/kinetic mechanism (23, 28). There are other acetyltransferases, including acetyl-

CoA:arylamine N-acetyltransferase from pigeon liver and N-hydroxyarylamine O-

acetyltransferase from Escherichia coli (29-30) that have been demonstrated to employ


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double displacement (ping-pong) mechanisms. Furthermore, bisubstrate analogues that

link acetyl-CoA to 7 or 20 amino acid peptides are poor p300 inhibitors, indirectly

arguing against a ternary complex mechanism (31).

p300/CBP acetylates all four core histone proteins (H2A, H2B, H3, and H4)

regardless of whether or not they are in the context of a nucleosome (21,22,32). In vitro,

p300 catalyzes the acetylation of the N-terminal tail of free histone H4 at K5, K8, K12,

and K16, all sites that are acetylated in vivo (21). However, in the context of a

nucleosome, p300 catalyzed acetylation of K5 and K8 is preferred (32).

In addition to histones, p300/CBP has been reported to acetylate a number of non-

histone proteins (reviewed in 2), including p53 (17, 33), GATA-1 (15,34), c-Myb (35),

E2F-1 (13,36), EKLF (12), ACTR, TIF2, SRC1 (16), AR (37), HMG I(Y) (38), HMG14

(39), Tat (40-41), TCF (42), TFIIE and TFIIF (43). Despite these findings, the molecular

determinants of p300 substrate selectivity are not established.


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MATERIALS AND METHODS Chemicals N-α-Fmoc-Cys(Boc-3-aminopropyl)OH, N-α-Fmoc-D-Lys(Boc)-OH, N-α-Fmoc-

Lys(Boc)(isopropyl)OH were from BACHEM (King of Prussia, PA). All other N-α-

Fmoc-amino acids were from Novabiochem (San Diego, CA). 14C-labelled acetyl-CoA

was from NEN (Boston, MA). Acetylated BSA, dithiothreitol, and Trizma-HCL were

from Sigma (St. Louis, MO).

Synthesis of N-α-Fmoc-6-hydroxy-norleucine N-α-Fmoc-6-hydroxy-norleucine (Fmoc-Hnl)(Figure 3) was synthesized by the

diazotization of N-α-Fmoc-L-lysine analogously to methods previously described (44).

N-α-Fmoc-L-lysine (1 g, 0.00271 moles) was dissolved in 4 mL of glacial acetic acid plus

0.7 mL of ddH2O and placed in an ice water bath in order to decrease the temperature of

the solution to below 10°C. To this solution, an aqueous solution (500 µL) of NaNO2

(4.1 mmoles, 281 mg, 1.5 equivalents) was added dropwise over 1 h. After addition of

the NaNO2 solution, the reaction was heated to 90°C for 20 minutes. The sample was

cooled to room temperature and concentrated in vacuo. At this stage, ddH2O (10 mL)

was added and the resulting slurry was extracted 4 times with methylene chloride (~10

mL) into which the desired product partitioned and this mixture was concentrated in

vacuo. The resultant oil was then dissolved in methanol and the desired product purified

by silica gel chromatography (3 x 15 cm) eluting with CH2Cl2:MeOH:32% aqueous acetic

acid (20:1:0.1). N-α-Fmoc-6-hydroxy-norleucine (Rf = 0.3) eluted from the column as a


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single spot and fractions containing the desired compound were pooled and concentrated.

The yield of N-α-Fmoc-6-hydroxy-norleucine was 15 % and the identity confirmed by

mass spectrometry and NMR.

N-Fmoc-6-hydroxy-norleucine. 1H NMR (CDCl3): δ 7.75 (d, J = 7.8 Hz, 2H), 7.60 (m,

2H), 7.39 (t, J = 7.4 Hz, 2H), 7.3 (m, 2H), 5.51 (d, J = 7.8 Hz, 1H), 4.38 (t, J = 7.4 Hz,

2H), 4.34 (m, 1H), 4.21 (t, J = 7.0 Hz, 1H), 3.10 (m, 2H), 1.80 (bm, 2H), 1.41-1.33 (m,

3.5H). ESIMS m/z 369.

Purification of Histone H4

An Escherichia coli overexpression plasmid encoding the gene for Xenopus lavis

histone H4 (a generous gift of T. Richmond) was used to over-produce histone H4.

Recombinant histone H4 was purified from inclusion bodies essentially as previously

described (45), except that a 2.6 x 90 cm Sephacryl 200 column (Amersham-Pharmacia

Biotech, Piscataway, NJ) was used for size exclusion chromatography and a 2.6 x 10 cm

SP sepharose column (Amersham-Pharmacia Biotech) was used for cation exchange

chromatography.

Histone H4 was refolded by dissolving 27.7 mg of purified protein in Unfolding

Buffer (7 M Guanidinium-HCl, 20 mM Tris-HCl pH 7.5, 10 mM dithiothreitol ) to a final

concentration of 2 mg/mL and incubated at room temperature for 15 minutes, at which

point the sample was diluted 2-fold with Unfolding Buffer, and incubated for a further 1

hour at room temperature. Histone H4 was dialyzed against 10 mM Tris-HCl pH 7.5, 250

mM NaCl, 1 mM EDTA, 5 mM β-mercaptoethanol for 1 hour at which point the dialysis

buffer was replaced and the sample dialyzed overnight. The following day the buffer was

again replaced and dialyzed for a further two hours. Triton X-100 was added to a final


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concentration of 0.1 % and the dialyzed sample concentrated using a 15 mL Centriprep

concentrator (Amicon) to a concentration of 4.3 mg/mL. MALDI mass spectra of pure

Histone H4 were collected at the JHMI Mass Spectrometry facility on a Voyager DE

MALDI-TOF workstation (Applied Biosystems). The mass spectrum was consistent with

the histone H4 (11224±11 m/z observed, 11236 m/z expected).

Synthesis and purification of peptide substrates

The peptides described herein were synthesized by automated solid phase peptide

synthesis using the Fmoc strategy on a Rainin PS3 machine. Briefly, the indicated N-α-

Fmoc amino acids were sequentially coupled to Wang resin linked to the C-terminal

residue of the desired peptide on a 0.1 mmol scale. Peptides were cleaved from the resin

with Reagent K (trifluoroacetic acid : phenol : H2O : thioanisole : ethanedithiol

(35:2:2:2:1) and subsequently precipitated with ice cold diethyl ether. Precipitates were

collected by centrifugation (3000 g, 10 minutes), the supernatants discarded, and the

pellets washed 2 times with cold diethyl ether (30 mL). Precipitated peptides were

dissolved in 5 mL of ddH2O, flash frozen and lyophilized. Peptides were purified by

reverse phase HPLC, as previously described (24). Electrospray mass spectrometry of

each peptide confirmed the correct structures.

Kinetic assay

Full-length human p300 protein was expressed in a baculovirus system and

purified as previously described (32). The HAT assay employed in this study has

essentially been described elsewhere (24). Briefly, the assay measures the production of


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14C-acetyl-peptide (or 14C-acetyl-protein ), a product of the p300 reaction, by separating

the components of the reaction on 16.5 % Tris-Tricine polyacrylamide gels, drying the

gels, and quantifying the radioactivity incorporated by phosphorimage analysis

(Molecular Dynamics). Generally, peptide and 14C-acetyl-CoA were pre-incubated in the

assay buffer (50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM dithiothreitol, and 50

µg/mL acetylated bovine serum albumin) for 10 minutes at 30 ° C, and 5 nM of full

length p300 was used to initiate the reaction. HAT activity was linear with respect to

time and enzyme concentration in the range employed. Unless otherwise noted the

concentration of 14C-acetyl-CoA was fixed at 20 µM (>5 Km) when measuring the steady-

state kinetic parameters for peptide or protein substrates. Assays were performed in

duplicate and these generally agreed within 20%. The initial rates obtained from these

assays were fit by non-linear least fit squares to Equation 1,

v = Vm[S]/(Km + [S]) (Equation 1), using the KaleidagraphTM version 3.5 software package. Initial velocity patterns

Initial velocity patterns for acetyl-CoA were obtained by determining the steady-

state kinetic parameters for acetyl-CoA at different fixed concentrations of the second

substrate, H4-20 (2, 3, 10 µM) or H4-12 (5, 7.5, 25 µM). The initial rates obtained were

fit to Equation 2,

v = (Vm[A][B])/(Kma[B] + Kmb[A] + [A][B]) (Equation 2),

using Kinetasyst IITM (IntelliKinetics), according to the algorithms of Cleland (46). Kma =

Km of acetyl-CoA; Kmb = Km of the peptide substrate.


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Dead-end analog inhibition studies

The initial rates derived from the dead-end analog inhibition experiments were fit

to equations representative of linear competitive inhibition (Equation 3), linear non-

competitive inhibition (Equation 4), or linear uncompetitive inhibition (Equation 5) by a

non-linear least fit squares approach using the Kinetasyst IITM software package:

v = Vm[S] / ([S] + Km(1 + [I]/Kis)) (Equation 3),

v = Vm[S] / ([S](1 + [I]/Kii) + Km(1 + [I]/Kis)) (Equation 4),

v = Vm[S] / ([S](1 + [I]/Kii) +Km) (Equation 5),

where Kii = Ki intercept, Kis = Ki slope. The best fits of the data were chosen through a

process of visual inspection, and a comparison of the standard errors and residuals

derived from fits of the data to Equations 3-5. The global fits obtained were used to

derive the lines drawn in double reciprocal plots. The two dead-end analogs used were

desulfo-CoA and H4-12-Arg peptide (N-RGRGGRGLGRGA-C). H4-12-Arg was used

as a dead-end analog of the H4-12 peptide, and as expected the H4-12-Arg peptide was

shown not to be a p300 substrate.

Solvent viscosity

The effect of solvent viscosity on the rate of p300 mediated acetylation of peptide

substrates was examined using the microviscogen sucrose (47-49). Acetyl-CoA (20 µM)

in combination with either H4-12 (120 µM), H4-12-Cap (2 mM), or H4-12-D-Lys (2

mM) were assayed with relative viscosity levels shown in Figure 5. No significant effect

on Km of either substrate was observed at the higher concentrations of sucrose used in this

study.


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Slow binding inhibition

Time course experiments were performed, in Assay Buffer, by incubating in 0.21

mL reaction volumes the H4-20 peptide (20 µM), 14C-acetyl-CoA (10 µM) , and p300 (5

nM) in the absence and presence of varying amounts (0, 75, 150, 300 nM) of the p300

specific inhibitor Lys-CoA (31). Portions (30 µL) of the reaction were withdrawn at

various time points, quenched with 6 µL of 6 x SDS-PAGE gel loading buffer, and

processed for Tris-Tricine polyacrylamide gel electrophoresis and phosphorimage

analysis, as previously described (24). Experiments were performed in duplicate at 30° C

and measured product formation did not exceed 20% of the available substrates.

Slopes for Vi (initial velocity), and Vs (steady-state velocity) were obtained by

linear regression of the two clearly observable phases of the time courses. Correlation

coefficients for these analyses were typically greater than 0.99. The reciprocals of the

values obtained for Vi and Vs were plotted against the concentration of Lys-CoA in order

to determine Ki and Ki*.

In order to measure an off rate (k6) for Lys-CoA, rapid dilution (400-fold) time

course experiments were performed. Reactions, H4-20 (20 µM) and 14C-acetyl-CoA (10

µM) in a total volume of 300 µL of Assay Buffer, were initiated by the addition of 0.75

µL of the p300• Lys-CoA inhibition complex (EI*), which had been pre-formed by

incubating p300 (1.2 µM) with Lys-CoA (5 µM) for 10 minutes at 30 ° C. At different

time points, 30 µL of the reaction was withdrawn and quenched with 6 µL of 6 x SDS-

PAGE gel loading buffer. Samples were processed for analysis as described above, and

the data obtained fit to Equation 6, using a non-linear least fit squares approach.


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P = vs(t - (1 – e-kt) / k6 (Equation 6)

P = nM product formed; vs = the steady-state rate; t = time; k6 = the first order off rate

constant; kt = k6 x t.


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RESULTS

p300 acetylation of histone H4 and peptide substrates -- In order to validate the use of

peptide substrates in catalytic mechanism studies on p300, we initially compared the

ability of this enzyme to acetylate protein as opposed to peptide substrates. We therefore

determined the steady-state kinetic parameters for pure recombinant histone H4 and a 21

amino acid peptide (H4-21) whose sequence is based on the N-terminal 21 amino acids of

human histone H4 (Table 1). The Km values for both peptide and full length histone H4

were both in the range of 1-3 µM and the kcats observed for both substrates were also

similar (0.5-0.8 s-1). The specificity constants (V/K) for the recombinant protein and the

H4-21 peptide were virtually identical, validating the use of the simpler peptide substrates

to further probe the catalytic mechanism of p300.

Since the determinants of the p300 interaction with histone H4 appear to be fully

present in the N-terminal region of this protein, the contributions made by different

regions of the peptide to substrate selectivity were determined by synthesizing a series of

smaller peptides. Since Lys-8 (histone H4 numbering) is a major in vivo site of

acetylation (21,32), these peptides were designed to maintain Lys-8 at a central position

while progressively shortening the sequence from both the N- and C-termini (Table 2).

For the H4-12, H4-15 and H4-17 peptides, the values of Km, kcat, and more importantly

V/K, are not much smaller than those obtained for the H4-21 peptide. The largest effect is

only a 6.5-fold decrease in the V/K of the H4-12 peptide, despite the loss of 9 amino acids

that could contribute to substrate selectivity. Thus, residues outside this 12 amino acid


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peptide, encompassing residues K-5 to K+6, are not critical for substrate recognition.

The H4-20 peptide, corresponding to the amino acid sequence of the H4-CoA-20

bisubstrate analogue (31), and the H4-20K8A peptide were also tested as substrates for the

p300 enzyme. The steady-state kinetic parameters for the H4-20 peptide are within 2-fold

of those obtained for the H4-21 peptide, whereas the parameters for the H4-20K8A are

modestly (6-fold) impaired.

p300 substrate specificity – In order to further define the requirements for substrate

selectivity, we examined the importance of the three remaining Lys residues by

synthesizing peptides in which these residues are replaced by Ala either individually or in

combination (Table 3). While values of the kcats observed for the H4-12K5A, H4-12K8A,

and H4-12K12A peptides are similar to the H4-12 peptide, the effect on V/K of each of the

individual changes is more significant, and ranges from 3.5- to 14.4-fold. To further

characterize the contributions of Lys residues to substrate specificity, a series of ‘double

mutant’ derivatives were synthesized: H4-12K5A/K8A, H4-12K5A/K12A, and H4-12K8A/K12A.

When these peptides were tested as p300 substrates (Table 3), no acetylation over the

assay background (V/K at least 300-fold reduced compared to the H4-12 peptide) was

observed indicating that the effect of these substitutions on substrate recognition is quite

dramatic. The H4-12K5R/K12A and the H4-12K5A/K12R peptides were synthesized to further

examine the contribution of positive charge, or hydrogen bonding, to substrate specificity

(Table 3). Both of these peptides were efficient p300 substrates, as the effects on V/K

were within ~3.2-fold of H4-12. In order to differentiate between the effects of

electrostatic interactions and hydrogen bonding on substrate recognition, the H4-


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12K5Cit/K12A peptide was synthesized. This peptide incorporates a citrulline (Cit; Figure 3)

in place of K5. No activity was observed above the assay background when the H4-

12K5Cit/K12A peptide was tested as a substrate for p300, indicating that an electrostatic

interaction at either the K-3 or K+4 position is critical for substrate recognition and

catalysis.

Novel lysine analogues – Using the H4-12K5R/K12A peptide as a minimal peptide substrate

(H4-12a), we probed the substrate specificity requirements of a target lysine itself, by

replacing the remaining Lys residue (Lys-8) with ornithine (Orn), 6-hydroxynorleucine

(Hnl), N-ε-isopropyl-lysine (Iso), 1-aminopropanol-cysteine (Cap), and D-lysine (D-Lys)

(Figure 3). Assay of peptides containing lysine analogues with p300 showed that all

demonstrated substantially decreased catalytic activity (Table 4). In fact, peptides with

Hnl and Iso showed no detectable activity. While both the H4-12a-Cap and H4-12a-D-

Lys peptides showed significant increases in Km, it cannot be determined at this stage

whether the primary defect was on binding or catalytic turnover. Acetyltransferase

activity with H4-12a-Orn peptide was detectable but severely reduced (~120-fold relative

to the H4-12K5R/K12A peptide), and accurate kcat and Km parameters could not be measured.

Two substrate kinetics – In an effort to better understand the catalytic mechanism of

p300, we determined the steady-state kinetic parameters for acetyl-CoA at various

concentrations of the H4-20 peptide. Double reciprocal plots of the initial velocities

obtained from these experiments exhibit a parallel line pattern (Figure 4) that is

suggestive of a ping-pong kinetic mechanism as opposed to a sequential (ternary


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complex) mechanism (50). A parallel line pattern was also observed in similar

experiments using the H4-12 peptide in place of H4-20 (data not shown). Intersecting

lines would have been expected for a sequential (ternary complex) mechanism. Initial

velocities were fit to Equation 2 and the kinetic parameters obtained are summarized in

Table 5.

Desulfo-CoA as a dead-end analogue inhibitor – Since desulfo-CoA lacks both an acetyl

group and a terminal sulfur atom it is expected to be a dead-end analog of acetyl-CoA.

Desulfo-CoA was shown to be a linear competitive inhibitor versus acetyl-CoA, whereas

this compound behaved as a linear uncompetitive inhibitor versus peptide substrate

(Table 6). These results are consistent with a ping-pong reaction mechanism.

H4-12-Arg as a dead-end analogue inhibitor – The H4-12-Arg peptide was evaluated as

a dead-end histone analogue. This peptide incorporates 3 Arg residues in place of the 3

Lys residues in the H4-12 peptide (K5, K8, and K12; histone H4 numbering), and as

expected was shown not to be a substrate for p300. The H4-12-Arg peptide was shown to

be a linear competitive inhibitor versus the H4-12 peptide and a linear uncompetitive

inhibitor of acetyl-CoA (Table 6), again consistent with a ping-pong kinetic mechanism

(50).

Solvent viscosity studies – The effect of solvent viscosity on the rate of the p300 catalyzed

reaction was examined using the microviscogen sucrose. In assays where the H4-12

peptide was the acetyl group acceptor a significant solvent viscosity effect on kcat was

noted (slope = +0.74 ± 0.10; Figure 5). However, only a small solvent viscosity effect


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was noted when the relatively ‘poor substrates’ (see above), H4-12a-Cap and H4-12a-D-

Lys, were assayed in this manner (slopes = +0.19 ± 0.03 and +0.12 ± 0.02, respectively),

suggesting that the diffusional release of one or more products is rate-limiting for p300

with a normal substrate, rather than the chemical step(s).

Lys-CoA is a slow binding p300 inhibitor – The kinetics of inhibition of the recently

described p300 selective inhibitor, Lys-CoA (Figure 6A; 31), were analyzed to gain

insight into the molecular basis underlying its potency. Initially, product formation

catalyzed by p300 as a function of time was examined in the presence of varying

concentrations of Lys-CoA (Figure 6B). These plots showed a decrease in velocity versus

time in a manner that is dependent on Lys-CoA concentration, typical of “slow, tight-

binding” behavior (51). After pre-formation of a Lys-CoA-p300 complex, a rapid-

dilution activity recovery time course was monitored (Figure 6C). These data fit well to a

first-order recovery process revealing that Lys-CoA slowly dissociates from p300, with a

rate constant (k6) of 0.15 min-1 (Scheme 1). Slow-binding inhibition is thought to result

when subsequent to the rather facile formation of the enzyme• inhibitor complex (E•I), a

tight-binding complex (E•I*) forms at a significantly lower rate due to a high

thermodynamic barrier (Scheme 1). Product formation can be described by Equation 7,

P = Vst + (Vo – Vs)((1 – e-kt)/k) (Equation 7),

where Vo = the initial rate; Vs = the steady-state rate; and k = the apparent first-order rate

constant for reaching the steady-state. Replots of 1/Vo and 1/Vs versus inhibitor

concentration (Figure 7) yield values for Ki and Ki* of 166 and 18.7 nM, respectively,


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which can be substituted into Equation 8 to determine the individual rate constant for the

formation of E•I* (k5). These data are summarized in Scheme 1.

Ki* / Ki = k6/(k5 + k6) (Equation 8)


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DISCUSSION The determination that HAT enzymes can acetylate a diverse array of non-histone

transcription-related factors heightens the importance of defining the mechanisms of

substrate binding and catalysis because such studies can aid in the identification of

potential substrates and help describe the molecular basis for the regulation of gene

expression. The rational design of enzyme inhibitors also requires a detailed

understanding of these processes, as the information gained can be incorporated into the

design to maximize the interactions between inhibitor and protein, thereby enhancing

potency. Therefore, to understand the p300 catalytic mechanism and substrate selectivity,

we initiated an in depth kinetic characterization of this enzyme.

Peptide substrates can be more attractive substrates than the natural protein

substrates for enzymatic studies because of their simplicity. Furthermore, the facile

incorporation of non-standard amino acids in synthetic peptides allows for increased

versatility in experimental analysis. The steady-state kinetic parameters for pure

recombinant histone H4 and a 21 amino acid peptide (H4-21) based on the amino

terminal sequence of this protein were similar as p300 substrates validating the

significance of further mechanistic studies on synthetic peptides. We next determined the

steady-state kinetic parameters for a number of smaller peptides, whose sequences are

also based upon the N-terminus of histone H4. The value of V/K observed for the H4-20

peptide is noteworthy because this peptide was used to simplify the synthesis of the H4-

CoA-20 bisubstrate analog, which has previously been shown to be a poor inhibitor of

p300 activity (31). The H4-20 peptide differs from the wild type histone H4 sequence by


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the replacement of His-18 with Asn. Since the H4-20 V/K is only 2-fold lower than the

H4-21 peptide, it is clear that the greater than 100-fold weaker inhibition observed with

H4-CoA-20 compared to Lys-CoA is not due to this amino acid substitution. Also

notable is the fact that, though Lys-8 is thought to be the preferred site of acetylation in

histone H4, p300 catalyzes the acetylation of H4-20K8A peptide fairly efficiently (21).

However, the lack of a more dramatic effect is not completely unexpected as p300 is

known to have a broad substrate specificity and can catalyze the acetylation of the lysines

5, 8, 12, and 16 in the N-terminal tail of histone H4 (21).

In an effort to identify histone H4 residues that contribute to substrate recognition,

we synthesized a series of smaller peptides that are 12, 15 and 17 amino acids in length.

Since the value of V/K for the H4-12 peptide is only decreased by 6.5-fold, it is clear that

the determinants of p300 substrate selectivity are largely located within a 12 amino acid

peptide. This is interesting since the H4-12 peptide is composed primarily of Gly (6/12)

and Lys (3/12) and lacks varied molecular determinants that can be critical for some

enzymes. Taking advantage of the simplicity afforded by this peptide substrate, we

examined the contribution of Lys-5, Lys-8, and Lys-12 to p300 recognition. Single

substitutions led to relatively modest declines (3- to 11-fold) in the V/Ks. In contrast,

kinetic analyses with the H4-12K5A/K8A, H4-12K5A/K12A, and H4-12K8A/K12A peptides

indicate that there is a preference for at least one positively charged or hydrogen bonding

residue, in addition to the target Lys, at either the K-3 or K+4 positions (Table 3). It

should be noted that the H4-12K8A peptide lacks a positively charged residue at either of

these positions, suggesting that longer range interactions may also in some cases

contribute to substrate recognition. The H4-12K5R/K12A and the H4-12K5A/K12R peptides


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were synthesized to substitute a non-acetylatable positively charged residue in place of

Lys residues at the 5 and 12 positions. Both of these peptides were efficiently acetylated.

In contrast, a peptide containing uncharged isosteric citrulline (H412a-Cit) is an

extremely poor substrate, indicating that positive charge rather than hydrogen-bonding

alone, is critical for p300 substrate recognition and catalysis.

To place the above peptide results into the context of potential physiological

protein substrates we manually aligned the amino acid sequences of known non-histone

substrates, centered around the primary acetylation sites (Figure 8). Interestingly, most of

the major acetylation sites (14/18) possess a positively charged residue at either the K-3

or K+4 position or both. With regard to those acetylation sites that do not contain such a

positive charge there are a number of potential explanations. First, these proteins may in

fact be poor p300 substrates. This appears to be the case for c-Myb, as this protein is

acetylated at a 10- to 100-fold lower rate than histone substrates (37). It is possible that

low processing efficiency is physiologically advantageous. Alternatively, positively

charged residues at the K+2 or K+3 positions may be able to substitute for a positively

charged residue at the K+4 position, as seems possible for both the androgen receptor and

HIV Tat. Further work will be needed to address this issue in more depth.

The determination that the H4-12K5R/K12A and the H4-12K5A/K12R peptides are p300

substrates helped to define a ‘minimal’ substrate containing only a single site capable of

being acetylated. One of these peptides, H4-12K5R/K12A, was used as a template to

characterize the structural requirements of the target Lys side chain for acetylation. Five

peptides that vary the position, steric accessibility, or the type of nucleophilic group in the

side chain were synthesized and their steady-state kinetic parameters determined.


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Overall, these structure-activity relationships highlight the stringent requirement of the

lysine structure, stricter in the degree of specificity than the GNAT enzyme serotonin N-

acetyltransferase shows for serotonin (47). It was noteworthy that H4-12a-D-Lys was

active as a substrate of p300. Many protein processing enzymes show an absolute

requirement for substrates with L-amino acids. While the 76-fold decrease in V/K for the

peptide containing D-Lys indicates that positioning of the ε-amino group is important for

substrate recognition, the fact that acetyl transfer is catalyzed at all shows unusual

plasticity on the part of p300. While it is theoretically possible that the acetylation could

be due to contaminating L-Lys containing peptides, the reduced solvent viscosity effect

for the H4-12a-D-Lys peptide compared to H4-12a argues against this possibility, as a

viscosity effect similar to that of the L-Lys containing peptide would have been predicted

in the contamination case.

Measurements of the solvent viscosity effect (SVE) on the rates of catalysis also

indicate that a diffusion-controlled step is at least partially rate-limiting for p300. To

ensure that the observed SVE was not due to a non-specific effect (eg. sucrose inhibition

of p300), the SVE was determined with the H4-12a-D-Lys and H4-12a-Cap peptides.

These poor substrates show only a small SVE, indicating that product release and/or a

related protein conformational change (as opposed to the chemical step(s)) is rate-limiting

for catalysis with standard lysine containing substrates. In contrast, the rate-limiting step

for the PCAF enzyme is the chemical step (24). The biological significance of this

finding is that it may suggest that allosteric activating molecules or post-translational

modifications could stimulate p300 by enhancing product release.


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The fact that bisubstrate analogues incorporating 7 or 20 amino acid peptide

conjugates are less potent p300 inhibitors than Lys-CoA suggested that this enzyme might

not obey a sequential kinetic mechanism (see above) (31). In a two substrate kinetic

analysis, the parallel line pattern observed in double reciprocal plots is consistent with a

ping-pong kinetic mechanism, arguing against a sequential mechanism which is seen in

HATs characterized previously (Scheme 2). The dead-end analogues desulfo-CoA and

H4-12-Arg afforded reciprocal patterns of competitive and uncompetitive inhibition, also

consistent with a ping-pong mechanism for p300. In future work it will be necessary to

identify and establish the kinetic competence of a possible covalent intermediate.

However, these findings argue against the previously stated proposal that all HATs will

show a conserved catalytic mechanism on the basis of structural studies on GNAT family

members and EsaI. Given p300’s unusually diverse role in biology, it can be proposed

that a ping-pong mechanism may allow broader substrate specificity since acetyl-CoA

and protein substrate do not have to line up simultaneously. Arylamine N-

acetyltransferase which shows a ping-pong mechanism also shows relatively broad

substrate specificity.

We have also examined the molecular basis for Lys-CoA inhibition of p300 and

shown that Lys-CoA is a potent slow-binding inhibitor of p300 with a dissociation rate

constant (t�� = 4.5 minutes) that is 116-fold lower than kcat (Scheme 1). These kinetics

suggest that a slow conformational change or solvent reorganization is necessary to

achieve the high affinity complex. Given the slow onset of the inhibition, care should be

taken in biological applications with this compound because initial p300 HAT dependent

processes will not be shut off immediately after addition of Lys-CoA.


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Conclusions

In summary, we have used in vitro p300 HAT assays to characterize the

determinants of p300 substrate selectivity, the kinetic mechanism of this enzyme, and the

molecular basis for the potent level of inhibition afforded by Lys-CoA. Through these

studies we have been able to define a minimal p300 peptide substrate and have

determined that efficient p300 substrate recognition requires a positively charged residue

at either the K-3 or K+4 positions. Furthermore, the determination that p300 employs a

ping-pong kinetic mechanism suggests that optimization of the CoASH/Lys linker may

afford inhibitors of even greater potency. Finally the results described herein shall form a

sound basis for the future study of the catalytic mechanism of p300.


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FIGURE LEGENDS Figure 1: Schematic representation of the p300 primary structure. Figure 2: Enzyme reaction catalyzed by p300. Figure 3: Structures of novel amino acids used in this study. Figure 4: Initial velocity patterns for p300. 1/Velocity versus 1/[acetyl-CoA] for the

p300 catalyzed reaction at fixed concentrations of the H4-20 peptide. Fixed

concentrations of peptide were 2 µM (solid squares), 3 µM (solid diamonds), and 10 µM

(solid triangles). The best fit of the data was to a ping-pong kinetic mechanism (Equation

2). For experimental details see “Materials and Methods”. Kinetic constants for this plot

are summarized in Table 5.

Figure 5: Effect of viscosity on the rate of the p300 catalyzed reaction. Plot of

kcat(control)/ kcat(viscogen) for various p300 peptide substrates versus relative viscosity. The

peptide substrates were H4-12 (open diamonds) (slope = 0.74 ± 0.10), H4-12a-Cap (open

circles) (slope = 0.19 ± 0.03), and H4-12a-D-Lys (open squares)(slope = 0.12 ± 0.02).

Figure 6: Lys-CoA is a slow-binding inhibitor of p300 activity. A. Structure of Lys-

CoA. B. Product formation versus time at various concentrations of Lys-CoA. Lys-CoA

concentrations were 0 µM (solid circles), 75µM (solid diamonds), 150 µM (solid

triangles), and 300 µM (open circles). See “Materials and Methods” for experimental

details. C. Time course experiments measuring product formation versus time after

rapid dilution of p300 (open squares) and pre-formed p300•Lys-CoA complex (solid

squares) to determine the rate constant for the dissociation of Lys-CoA from p300.


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Figure 7: Replots of 1/Vo and 1/Vs versus the concentration of Lys-CoA. The

reciprocals of the slopes obtained from Figure 6, representing the initial rate (Vo) (open

squares) and the steady-state rate Vs (open circles), were plotted versus the concentration

of Lys-CoA.

Figure 8: Alignment of known non-histone p300 substrates. Primary sequences were

manually aligned to position the major sites of acetylation in a central location. Certain

proteins have multiple entries because these proteins are multiply acetylated by p300.

Bolded K’s identify the sites of acetylation and also identify positively charged residues

at either the K-3 or K+4 positions. Sites of acetylation have been previously documented:

dTCF (41), hACTR (16), cGATA-1 (33), mGATA-1 (15), hE2F1 (13), hHMG14 (38),

hHMGI(Y) (37), p53 (17,32), hEKLF (12), androgen receptor (36), Tat (39-40), c-myb

(34).

Scheme 1: Slow-binding inhibition.

Scheme 2: Ping Pong kinetic mechanism.


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TABLES

Table 1: p300 steady-state kinetic parameters for protein and peptide substrates. Substrate Km

(µM) kcat (s-1)

V/K (s-1M-1)

Histone H4 2.46±0.41 0.76±0.03 3.1 x 105

H4-21 1.52±0.54 0.46±0.06 3.0 x 105

AcCoA1 1.24±0.19 0.26±0.01 2.1 x 105

1. The kinetic parameters for acetyl CoA were determined in the presence of the H4-20 peptide (25 µM).

Table 2: p300 steady-state kinetic parameters for 12 to 21 amino acid peptide substrates.

Peptide Peptide Sequence Km

(µM) kcat

(s-1)

V/K

(M-1s-1)

H4-12 RGKGGKGLGKGA 8.3±1.4 0.38±0.01 4.6x104 H4-15 GRGKGGKGLGKGGAK 4.0±0.5 0.40±0.01 1.0x105 H4-17 SGRGKGGKGLGKGGAKA 7.8±0.6 0.39±0.01 5.0x104 H4-20 SGRGKGGKGLGKGGAKRNRA 1.9±0.4 0.29±0.02 1.5x105 H4-20K8A SGRGKGGAGLGKGGAKRNRA 9.6±2.2 0.19±0.01 2.0x104 H4-21 SGRGKGGKGLGKGGAKRHRKV 1.5±0.5 0.46±0.06 3.0x105


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Table 3: The contribution of positive charge to substrate selectivity. Peptide Peptide Sequence Km

(µM) kcat

(s-1)

V/K

(M-1s-1)

H4-12 RGKGGKGLGKGA 8.3±1.4 0.38±0.01 4.6x104 H4-12K5A RGAGGKGLGKGA 87±10 0.28±0.01 3.2x103 H4-12K8A RGKGGAGLGKGA 96±20 0.46±0.03 4.8x103 H4-12K12A RGKGGKGLGAGA 40±5.0 0.53±0.02 1.3x104 H4-12K5A/K8A RGAGGAGLGKGA -- <0.016±0.001 <16 H4-12K5A/K12A RGAGGKGLGAGA -- ≤3.8x10-3±3x10-5 ≤3.8 H4-12K8A/K12A RGKGGAGLGAGA -- <0.010±0.001 <10 H4-12K5R/K12A RGRGGKGLGAGA 17±2.4 0.34±0.01 2.0x104 H4-12K5A/K12R RGAGGKGLGRGA 49±9.0 0.71±0.04 1.4x104 H4-12K5Cit/K12A RGXGGKGLGAGA -- ≤5.9x10-3 ± 9x10-4 ≤5.9 X = citrulline.

Table 4: p300 steady-state kinetic parameters for novel lysine analogues. Kinetic parameters were determined according to “Materials and Methods” with saturating concentrations (20 µM) of acetyl-CoA.

Peptide Km

(µM) kcat

(s-1)

V/K

(M-1s-1)

H4-12a1 17±2.4 0.34±0.01 2.0x104 H4-12a -Orn -- ≤2.9x10-3 ± 5.8x10-5 ≤2.9 H4-12 a-Hnl -- <4.4x10-4 ± 8.0x10-7 <4.4 H4-12a-Iso -- <6.3x10-4 ± 1.4x10-4 <6.3 H4-12 a-Cap 756±194 0.15±0.02 200 H4-12 a-D-Lys 723±120 0.19±0.02 260 1. The peptides listed in this table are derivatives of the H4-12K5R/K12A peptide and such derivatives are referred to as H4-12a.


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Table 5: Ping-Pong kinetic parameters. See Figure 4 for experimental details. Kma = Km acetyl-CoA; Kmb = Km peptide Fixed substrate

kcat (s-1)

Kma

(µM) Kmb

(µM)

H4-12 0.54±0.10 5.0±1.3 25.1±6.4 H4-20 1.15±0.28 10.9±3.1 26.0±7.5 Table 6: Dead-end inhibition of p300 Inhibitor Varied

substrate

Constant substrate

Inhibition pattern

Kis (µM)

Kii

(µM)

Desulfo-CoA1 Acetyl-

CoA H4-20 (25 µM) competitive 8.07±0.59 --

Desulfo-CoA2 H4-12 Acetyl-CoA (20 µM)

uncompetitive -- 71.7±9.8

H4-12-Arg3,4

H4-12 Acetyl-CoA (20 µM)

competitive 178±45 --

H4-12-Arg2

Acetyl- CoA

H4-12 (40 µM) uncompetitive -- 548±70

1. Inhibition experiments were performed at 0, 50, and 100 µM desulfo-CoA. 2. Inhibition experiments were performed at 0, 500, and 1000 µM of the peptide substrate. 3. Inhibition experiments were performed at 0, 400, and 800 µM of the peptide substrate. 4. The H4-12-Arg peptide is a non-acetylatable analogue of the H4-12 peptide with Lys residues replaced with Arg. The sequence of this peptide is: NH2-RGRGGRGLGRGA-COO-.


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Paul R. Thompson, Hisanori Kurooka, Yoshihiro Nakatani and Philip A. Coleacetyltransferase activity

Transcriptional coactivator protein p300: Kinetic characterization of its histone

published online July 9, 2001J. Biol. Chem.

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