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J. Biol. Chem., Vol. 276, Issue 36, 33721-33729, September 7, 2001

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Transcriptional Coactivator Protein p300

KINETIC CHARACTERIZATION OF ITS HISTONE ACETYLTRANSFERASE ACTIVITY^*

Paul R. Thompson, Hisanori Kurooka^§, Yoshihiro Nakatani^§, and Philip A. Cole^¶

From the  Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, Baltimore, Maryland 21205 and the ^§ Dana-Farber Cancer Institute, Boston, Massachusetts 02115

Received for publication, May 23, 2001, and in revised form, July 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

The p300/cAMP response element-binding protein-binding protein (CBP) family members include human p300 and cAMP response element-binding protein-binding protein, which are both important transcriptional coactivators and histone acetyltransferases. Although the role of these enzymes in transcriptional regulation has been extensively documented, the molecular mechanisms of p300 and CBP histone acetyltransferase 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

Since the initial revelation that yeast Gcn5p (Gcn = global control non-repressed) is a histone acetyltransferase (HAT)¹ (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 Refs. 2-5). More than 20 HATs have now been identified and these have been classified into one of five families (GNAT, MYST (MOZ, YBF2/Sas3, Sas2, Tip60), TAF_II250, nuclear receptor coactivators, and p300/cAMP response element-binding protein-binding protein (CBP)), primarily based upon amino acid sequence homology (2).

The p300/CBP family of HATs is represented by two proteins, p300 and 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 cAMP response element-binding protein 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 polymerase II holoenzyme, transcription factors, and nuclear hormone receptors and their co-activators (2, 10-19) (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the p300 primary structure.

Following the determination that 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 (Fig. 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 (24-28).

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Enzyme reaction catalyzed by p300.

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 family member Esa1 (for 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 (27). 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, 27). 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 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 Lys-5, Lys-8, Lys-12, and Lys-16, all sites that are acetylated in vivo (21). However, in the context of a nucleosome, p300-catalyzed acetylation of Lys-5 and Lys-8 is preferred (32).

In addition to histones, p300/CBP has been reported to acetylate a number of non-histone proteins (reviewed in Ref. 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), and TFIIE and TFIIF (43). Despite these findings, the molecular determinants of p300 substrate selectivity are not established.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

Chemicals-- N--Fmoc-Cys(Boc-3-aminopropyl)OH, N--Fmoc-D-Lys(Boc)-OH, N--Fmoc-Lys(Boc)(isopropyl)OH were from Bachem (King of Prussia, PA). All other N--Fmoc-amino acids were from Novabiochem (San Diego, CA). ¹⁴C-Labeled acetyl-CoA was from PerkinElmer Life Sciences. Acetylated bovine serum albumin, dithiothreitol, and Trizma-HCl were from Sigma.

Synthesis of N--Fmoc-6-hydroxynorleucine-- N--Fmoc-6-hydroxynorleucine (Fmoc-Hnl) (Fig. 3) was synthesized by the diazotization of N--Fmoc-L-lysine analogously to methods described previously (44). N--Fmoc-L-lysine (1 g, 0.00271 mol) 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 mmol, 281 mg, 1.5 eq) was added dropwise over 1 h. After addition of the NaNO₂ solution, the reaction was heated to 90 °C for 20 min. 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 four 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 × 15 cm) eluting with CH₂Cl₂:MeOH:32% aqueous acetic acid (20:1:0.1). N--Fmoc-6-hydroxynorleucine (R_F = 0.3) eluted from the column as a single spot, and fractions containing the desired compound were pooled and concentrated. The yield of N--Fmoc-6-hydroxynorleucine was 15%, and the identity was confirmed by mass spectrometry and NMR.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Structures of novel amino acids used in this study.

N-Fmoc-6-hydroxynorleucine-- ¹H NMR (CDCl₃):  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). Electrospray ionization-mass spectrometry m/z was 369.

Purification of Histone H4-- An E. coli overexpression plasmid encoding the gene for Xenopus laevis histone H4 (a generous gift of T. Richmond) was used to overproduce histone H4. Recombinant histone H4 was purified from inclusion bodies essentially as described previously (45), except that a 2.6 × 90-cm Sephacryl 200 column (Amersham Pharmacia Biotech) was used for size exclusion chromatography and a 2.6 × 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 min, at which point the sample was diluted 2-fold with Unfolding Buffer, and incubated for another 1 h at room temperature. Histone H4 was dialyzed against 10 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM EDTA, 5 mM -mercaptoethanol for 1 h, at which point the dialysis buffer was replaced and the sample dialyzed overnight. The following day the buffer was again replaced and dialyzed for another 2 h. Triton X-100 was added to a final concentration of 0.1% and the dialyzed sample concentrated using a 15-ml Centriprep concentrator (Amicon) to a concentration of 4.3 mg/ml. Matrix-assisted laser desorption ionization mass spectra of pure histone H4 were collected at the JHMI Mass Spectrometry facility on a Voyager DE matrix-assisted laser desorption ionization/time of flight work station (Applied Biosystems). The mass spectrum was consistent with the histone H4 (11,224 ± 11 m/z observed, 11,236 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--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 min), the supernatants discarded, and the pellets washed two 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 high performance liquid chromatography, as described previously (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 described previously (32). The HAT assay employed in this study has essentially been described elsewhere (24). Briefly, the assay measures the production of [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 min at 30 °C, and 5 nM 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 K_m) 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, using the Kaleidagraph^TM version 3.5 software package.

(Eq. 1)

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, and 10 µM) or H4-12 (5, 7.5, and 25 µM). The initial rates obtained were fit to Equation 2, using Kinetasyst II^TM (IntelliKinetics), according to the algorithms of Cleland (46).

(Eq. 2)

K_ma = K_m of acetyl-CoA; K_mb = K_m of the peptide substrate.

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 II^TM software package.

(Eq. 3)

(Eq. 4)

(Eq. 5)

K_ii = K_i intercept, K_is = K_i 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; 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 H4-12 (120 µM), H4-12-Cap (2 mM), or H4-12-D-Lys (2 mM) were assayed with relative viscosity levels shown in Fig. 5. No significant effect on K_m of either substrate was observed at the higher concentrations of sucrose used in this study.

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, and 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× SDS-polyacrylamide gel electrophoresis gel loading buffer, and processed for Tris-Tricine polyacrylamide gel electrophoresis and phosphorimage analysis, as described previously (24). Experiments were performed in duplicate at 30 °C, and measured product formation did not exceed 20% of the available substrates.

Slopes for V_i (initial velocity), and V_s (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 V_i and V_s were plotted against the concentration of Lys-CoA in order to determine K_i and K_i*.

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 min at 30 °C. At different time points, 30 µl of the reaction was withdrawn and quenched with 6 µl of 6× SDS-polyacrylamide gel electrophoresis 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.

(Eq. 6)

P = nM product formed; v_s = the steady-state rate; t = time; k₆ = the first order off rate constant; kt = k₆ × t.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

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), the sequence of which is based on the N-terminal 21 amino acids of human histone H4 (Table I). The K_m values for both peptide and full-length histone H4 were both in the range of 1-3 µM, and the k_cat values observed for both substrates were also similar (0.5-0.8 s¹). 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 II). For the H4-12, H4-15, and H4-17 peptides, the values of K_m, k_cat, 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 peptide, encompassing residues Lys-5 to +Lys-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 H4-20^K8A 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 H4-20^K8A 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 III). Although the k_cat values observed for the H4-12^K5A, H4-12^K8A, and H4-12^K12A 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: H4-12^K5A/K8A, H4-12^K5A/K12A, and H4-12^K8A/K12A. When these peptides were tested as p300 substrates (Table III), no acetylation over the assay background (V/K at least 300-fold reduced compared with the H4-12 peptide) was observed, indicating that the effect of these substitutions on substrate recognition is quite dramatic. The H4-12^K5R/K12A and the H4-12^K5A/K12R peptides were synthesized to further examine the contribution of positive charge, or hydrogen bonding, to substrate specificity (Table III). 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 H4-12^K5Cit/K12A peptide was synthesized. This peptide incorporates a citrulline (Cit; Fig. 3) in place of K5. No activity was observed above the assay background when the H4-12^K5Cit/K12A peptide was tested as a substrate for p300, indicating that an electrostatic interaction at either the Lys-3 or +Lys-4 position is important for substrate recognition and catalysis.

Novel Lysine Analogues-- Using the H4-12^K5R/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--isopropyl-lysine, 1-aminopropanol-cysteine (Cap), and D-lysine (D-Lys) (Fig. 3). Assay of peptides containing lysine analogues with p300 showed that all demonstrated substantially decreased catalytic activity (Table IV). In fact, peptides with Hnl and N--isopropyl-lysine showed no detectable activity. Although both the H4-12a-Cap and H4-12a-D-Lys peptides showed significant increases in K_m, 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 H4-12^K5R/K12A peptide), and accurate k_cat and K_m 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 (Fig. 4) that is suggestive of a ping-pong kinetic mechanism as opposed to a sequential (ternary 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 V.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Initial velocity patterns for p300. Figure shows 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 V.

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 VI). 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 (Lys-5, Lys-8, and Lys-12; 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 VI), 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 k_cat was noted (slope = +0.74 ± 0.10; Fig. 5). However, only a small solvent viscosity effect 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).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of viscosity on the rate of the p300-catalyzed reaction. Figure shows 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).

Lys-CoA Is a Slow Binding p300 Inhibitor-- The kinetics of inhibition of the recently described p300-selective inhibitor, Lys-CoA (Fig. 6A; Ref. 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 (Fig. 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 (Fig. 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 min¹ (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.

View larger version (13K): [in this window] [in a new window]   Fig. 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), 75 µM (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.

View larger version (9K): [in this window] [in a new window]   Scheme 1.   Slow binding inhibition.

(Eq. 7)

V_o = the initial rate; V_s = the steady-state rate; and k = the apparent first-order rate constant for reaching the steady-state. Replots of 1/V_o and 1/V_s versus inhibitor concentration (Fig. 7) yield values for K_i and K_i* of 166 and 18.7 nM, respectively, 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.

(Eq. 8)

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Replots of 1/Vo and 1/Vs versus the concentration of Lys-CoA. The reciprocals of the slopes obtained from Fig. 6, representing the initial rate (Vo) (open squares) and the steady-state rate Vs (open circles), were plotted versus the concentration of Lys-CoA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

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 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 with Lys-CoA is not due to this amino acid substitution. Also notable is the fact that, although Lys-8 is thought to be the preferred site of acetylation in histone H4, p300 catalyzes the acetylation of H4-20^K8A 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 (3-11-fold) in the V/Ks. In contrast, kinetic analyses with the H4-12^K5A/K8A, H4-12^K5A/K12A, and H4-12^K8A/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 Lys-3 or  Lys-4 positions (Table III). It should be noted that the H4-12^K8A 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 H4-12^K5R/K12A and the H4-12^K5A/K12R peptides 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 (Fig. 8). Interestingly, most of the major acetylation sites (14/18) possess a positively charged residue at either the Lys-3 or +Lys-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 10-100-fold lower rate than histone substrates (37). It is possible that low processing efficiency is physiologically advantageous. Alternatively, positively charged residues at the +Lys-2 or +Lys-3 positions may be able to substitute for a positively charged residue at the +Lys-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.

View larger version (57K): [in this window] [in a new window]   Fig. 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. Boldface K identifies the sites of acetylation and also identify positively charged residues at either the Lys-3 or +Lys-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), and c-Myb (34).

The determination that the H4-12^K5R/K12A and the H412^K5A/K12R peptides are p300 substrates helped to define a "minimal" substrate containing only a single site capable of being acetylated. One of these peptides, H4-12^K5R/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. 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. Although the 76-fold decrease in V/K for the peptide containing D-Lys indicates that positioning of the -amino group is important for substrate recognition, the fact that acetyl transfer is catalyzed at all shows unusual plasticity on the part of p300. Although 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 with 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 nonspecific effect (e.g. 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 p300/CBP-associated factor 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.

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 Esa1. Given the unusually diverse role of p300 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 that shows a ping-pong mechanism also shows relatively broad substrate specificity.

View larger version (8K): [in this window] [in a new window]   Scheme 2.   Ping-pong kinetic mechanism.

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_1/2 = 4.5 min) that is 116-fold lower than k_cat (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.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

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 Lys-3 or +Lys-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.

	FOOTNOTES

^* This work was supported in part by grants from the Canadian Institutes of Health Research (to P. R. T.) and the National Institutes of Health (to P. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Sciences, The Johns Hopkins University, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-0540; E-mail: pcole@jhmi.edu.

Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M104736200

	ABBREVIATIONS

The abbreviations used are: HAT, histone acetyltransferase; CBP, cAMP response element-binding protein-binding protein; Cit, citrulline; Orn, ornithine; Hnl, 6-hydroxynorleucine; Cap, 1-aminopropanol-cysteine; D-Lys, D-lysine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; Boc, butoxycarbonyl; ddH₂O, double-distilled H₂O; SVE, solvent viscosity effect; GNAT, GCN-5-related N-acetyltransferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

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