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• W1963753695 abstract "The transcriptional coactivator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for activation of target genes. GCN5 is a member of a large family of histone acetyltransferases that are conserved between yeast and humans. To understand the molecular mechanisms of histone/protein acetylation, a detailed kinetic analysis was performed. Bi-substrate kinetic analysis using acetyl-coenzyme A (AcCoA) and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis. Product inhibition studies revealed that the product CoA was a competitive inhibitor versus AcCoA. Desulfo-CoA, a dead-end inhibitor, also demonstrated simple competitive inhibition versus AcCoA. Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA. These results support a sequential ternary complex (ordered Bi-Bi) kinetic mechanism, where AcCoA binds first, followed by H3 histone. Acetylated (Lys14Ac) H3 product is released first, and CoA is the last product to leave. Also, two methods were developed to measure the binding affinities of AcCoA/CoA for GCN5. Employing the fluorescent CoA analog etheno-CoA (εCoA, 1-N 6-etheno-CoA), aK d for εCoA of 5.1 ± 1.1 μmwas determined by fluorescence anisotropy. This value was similar to the K d value of 8.5 ± 2.6 μmfor AcCoA obtained using equilibrium dialysis and to theK i (inhibition constant) of 6.7 μmfor CoA obtained from steady-state kinetic assays. Together, these data suggest that the acetyl moiety of AcCoA contributes little to the binding energy. The transcriptional coactivator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for activation of target genes. GCN5 is a member of a large family of histone acetyltransferases that are conserved between yeast and humans. To understand the molecular mechanisms of histone/protein acetylation, a detailed kinetic analysis was performed. Bi-substrate kinetic analysis using acetyl-coenzyme A (AcCoA) and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis. Product inhibition studies revealed that the product CoA was a competitive inhibitor versus AcCoA. Desulfo-CoA, a dead-end inhibitor, also demonstrated simple competitive inhibition versus AcCoA. Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA. These results support a sequential ternary complex (ordered Bi-Bi) kinetic mechanism, where AcCoA binds first, followed by H3 histone. Acetylated (Lys14Ac) H3 product is released first, and CoA is the last product to leave. Also, two methods were developed to measure the binding affinities of AcCoA/CoA for GCN5. Employing the fluorescent CoA analog etheno-CoA (εCoA, 1-N 6-etheno-CoA), aK d for εCoA of 5.1 ± 1.1 μmwas determined by fluorescence anisotropy. This value was similar to the K d value of 8.5 ± 2.6 μmfor AcCoA obtained using equilibrium dialysis and to theK i (inhibition constant) of 6.7 μmfor CoA obtained from steady-state kinetic assays. Together, these data suggest that the acetyl moiety of AcCoA contributes little to the binding energy. histone acetyltransferases yeast GCN5 acetyl-coenzyme A 1-N 6-etheno-CoA The histone acetyltransferase (HAT)1 GCN5 fromSaccharomyces cerevisiae catalyzes the transfer of an acetyl moiety from acetyl-CoA (AcCoA) to the ε-amino group of lysine 14 of histone H3 (Scheme 1). GCN5 was originally identified as a transcriptional activator that was necessary to promote maximal levels of GCN4-dependent transcription (1.Georgakopoulos T. Thireos G. EMBO J. 1992; 11: 4145-4152Crossref PubMed Scopus (255) Google Scholar). AcCoA+histone H3 ⇄yGCN5 CoA+acetylated histone H3 SCHEME 1Recent reports have demonstrated that the acetylation of specific lysine side chains in the amino termini of core histones plays a crucial role in the transcriptional activation of specific target genes (2.Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (468) Google Scholar, 3.Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 4.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2401) Google Scholar). The yeast GCN5 (yGCN5) HAT shows a strong preference for acetylation of lysine 14 of histone H3 in vitro with reported broader specificity in vivo. In vivo yGCN5 has been reported to acetylate lysine 9 and 18 of histone H3, residues 8 and 16 of histone H4, and lysine residues in the amino-terminal tail of histone H2B, albeit to a much lesser degree than lysine 14 of histone H3 (5.Grant P.A. Eberharter A. John S. Cook R.G. Turner B.M. Workman J.L. J. Biol. Chem. 1999; 274: 5895-5900Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 6.Zhang W. Bone J.R. Edmondson D.G. Turner B.M. Roth S.Y. EMBO J. 1998; 17: 3155-3167Crossref PubMed Scopus (273) Google Scholar). Models have been proposed in which these site-specific acetylations in the amino termini of histones lead to altered nucleosomal chromatin structure by disrupting histone-DNA contacts and histone-histone contacts (7.Hansen J.C. Tse C. Wolffe A.P. Biochemistry. 1998; 37: 17637-17641Crossref PubMed Scopus (216) Google Scholar). Enrichment of acetylation on specific lysine residues suggests that differential acetylation within distinct chromatin loci may play a key role in transcriptional regulation (2.Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (468) Google Scholar, 3.Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 4.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2401) Google Scholar). Several classes of HATs have been identified: p300/CBP (8.Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar) cytosolic HAT 1 (9.Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Cell. 1998; 94: 427-438Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), P/CAF (10.Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), TAFII250 (11.Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar), and SRC-1 (12.Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1066) Google Scholar). Among HAT enzymes, yGCN5, which is a P/CAF family member, has been the most thoroughly characterized. yGCN5 is typically found in two high molecular mass complexes, SAGA (1.8 MDa) and Ada (0.8 MDa). The SAGA complex activates transcription by association with acidic activation domains of various transcription factors and results in the acetylation of nucleosomal histones H3 and H2B (13.Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (882) Google Scholar). The Ada complex has also been shown to acetylate nucleosomal histone H3 and histone H2B (14.Eberharter A. Sterner D.E. Schieltz D. Hassan A. Yates J.R.R. Berger S.L. Workman J.L. Mol. Cell. Biol. 1999; 19: 6621-6631Crossref PubMed Scopus (149) Google Scholar). Recently, several crystallographic and nuclear magnetic resonance (NMR) molecular models have been solved for the catalytic domains of yGCN5, P/CAF, and a Tetrahymena homologue (p55) of yGCN5 (15.Clements A. Rojas J.R. Trievel R.C. Wang L. Berger S.L. Marmorstein R. EMBO J. 1999; 18: 3521-3532Crossref PubMed Scopus (138) Google Scholar, 16.Rojas J.R. Trievel R.C. Zhou J. Mo Y. Li X. Berger S.L. Allis C.D. Marmorstein R. Nature. 1999; 401: 93-98Crossref PubMed Scopus (234) Google Scholar, 17.Lin Y. Fletcher C.M. Zhou J. Allis C.D. Wagner G. Nature. 1999; 400: 86-89Crossref PubMed Scopus (90) Google Scholar). These structures demonstrated that the high degree of sequence homology between these enzymes is manifested by a similar overall structure. The binary complex of P/CAF·CoA and the ternary complex ofTetrahymena p55·CoA·H3 peptide revealed that residues contacting CoA in the active site are highly conserved. In addition, a biochemical study (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) of yGCN5 enzymatic activity implicated the conserved glutamic acid 173 as a general base catalyst in the GCN5 HAT reaction, deprotonating the ε-amino group of lysine 14 in histone H3. Elucidating the mechanism of HAT catalysis will provide a basis for understanding the link between histone acetylation and gene activation. In this study we have systematically determined the overall kinetic mechanism, developed methods for measuring substrate/product binding affinities (fluorescence anisotropy and equilibrium dialysis), and determined the order of substrate binding and the order of product release by employing the product inhibitors CoA and acetylated (Lys14Ac) H3 peptide and the dead-end inhibitor desulfo-CoA. The results are consistent with a fully ordered Bi-Bi kinetic mechanism, where AcCoA is the first substrate to bind, and CoA is the last product released. All chemicals were of the highest grade commercially available. Histone H3 peptide, ARTKQTARKSTGGKAPPKQLC, and the corresponding acetylated H3 peptide (Lys14Ac), corresponding to the 20 amino-terminal residues of human histone H3 and an additional carboxyl-terminal cysteine, were synthesized by the Protein Chemistry Core Lab at The Baylor College of Medicine. Acetyl-CoA was purchased from Roche Molecular Biochemicals. Calf thymus histones were purchased from Calbiochem. [3H]Acetyl-CoA (1.88 Ci/mmol) was from NEN Life Sciences Products. P81 phosphocellulose disks were from Life Technologies, Inc. Dispo-Equilibrium Dialyzers were from Amika Corp. εCoA, 1-N 6-etheno-CoA was from Sigma. All other reagents were from Sigma or Fisher. The catalytic domain (amino acids 99–262) of yGCN5 was recombinantly expressed by isopropyl-B- d-thiogalactopyranoside induction for 12 h at 25 °C and purified from BL21-DE3 bacteria as described previously (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19.Kim Y. Tanner K.G. Denu J.M. Anal. Biochem. 2000; 280: 308-314Crossref PubMed Scopus (83) Google Scholar). After cation exchange chromatography on S-Sepharose, fractions (assessed by SDS-polyacrylamide gel electrophoresis) were pooled and concentrated and then subjected to size-exclusion chromatography on G-75 Sephadex. Purity was analyzed by SDS-PAGE, and fractions were concentrated and stored at −20 °C until use. Protein concentrations were determined by the method of Bradford (20.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). yGCN5 histone acetyltransferase activity was monitored continuously using a Multiskan Ascent microplate reader (LabSystems, Franklin, MA) as described previously (19.Kim Y. Tanner K.G. Denu J.M. Anal. Biochem. 2000; 280: 308-314Crossref PubMed Scopus (83) Google Scholar). Briefly, the CoASH generated in the HAT reactions was continuously measured by using a coupled enzyme system with pyruvate dehydrogenase. The CoASH-dependent oxidation of pyruvate was accompanied by the reduction of NAD to NADH, which was measured spectrophotometrically at 340 nm (ε340NADH = 6220m−1cm−1). The HAT assay reaction mixtures (70 μl) contained 0.2 mm NAD, 0.2 mm thiamine pyrophosphate, 5 mm MgCl2, 1 mmdithiothreitol, 2.4 mm pyruvate, and 0.03 units of pyruvate dehydrogenase (1 unit of dehydrogenase is defined by the manufacturer (Sigma) to be the conversion of 1.0 μmol of β-NAD to β-NADH/min at pH 7.4 at 30 °C). The assay buffer was 100 mm sodium acetate, 50 mm Bis-Tris, and 50 mm Tris and pH 7.5. All assays were performed at 25 °C and were initiated by the addition of yGCN5. The rates were analyzed continuously for up to 5 min, and background rates resulting from the spontaneous formation of CoA were subtracted from the initial velocities derived from the yGCN5-catalyzed reactions. Under these conditions, the coupled enzyme reaction does not limit the observed initial velocities (19.Kim Y. Tanner K.G. Denu J.M. Anal. Biochem. 2000; 280: 308-314Crossref PubMed Scopus (83) Google Scholar). Also, only the linear portion of the kinetic traces were used to determine the initial rates. These were typically linear for up to 5 min. Stock solutions of the H3 peptide or acetylated (Lys14Ac) H3 peptide were prepared fresh daily in the presence of 5 mmdithiothreitol. Alternatively, yGCN5 activity was measured using [3H]AcCoA, the P81-filter binding assay as described previously (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The Bi-substrate kinetic analysis was performed at AcCoA concentrations spanning 1–20 μm and H3 peptide or calf thymus histone concentrations spanning 25–730 μm. The yGCN5 HAT activity was monitored via the coupled-enzyme spectrophotometric assay using either pyruvate dehydrogenase or α-ketoglutarate dehydrogenase. The data were fitted to three possible kinetic mechanisms: the sequential (ternary complex) mechanism equation (Equation 1), the ping-pong (covalent intermediate) mechanism equation (Equation 2), and the equilibrium-ordered equation (Equation 3), using the algorithms of Cleland (21.Cleland W.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1977; 45: 273-387PubMed Google Scholar) and the computer program KinetAsyst (IntelliKinetics, State College, PA) and a nonlinear least squares analysis. v=Vm×[A]×[B]/((Kia×Kb)+(Kma×[B])Equation 1 +(Kmb×[A])+([A]×[B])) v=Vm×[A]×[B]/((Ka×[B])+(Kb×[A])+([A]×[B]))Equation 2 v=Vm×[A]×[B]/((Ka×Kb)+(Kb×[A])+([A]×[B]))Equation 3 The product inhibitors CoA and acetylated (Lys14Ac) H3 peptide, as well as the substrate analog desulfo-CoA (lacking only the terminal sulfhydryl of CoA) were used in steady-state inhibition studies. To investigate whether competitive (Equation 4), noncompetitive (Equation 5), or uncompetitive (Equation6) inhibition was observed, the data were fitted to the respective inhibition equations based on the algorithms defined by Cleland (21.Cleland W.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1977; 45: 273-387PubMed Google Scholar) using a nonlinear least squares analysis. v=Vm×[S]/[Km(1+I/[Kis)+[S]]Equation 4 v=Vm×[S]/[Km(1+I/Kis)+[S](1+I/Kii)]Equation 5 v=Vm×[S]/[Km+[S](1+I/Kii)]Equation 6 Equilibration was performed using Dispo-Equilibrium Dialyzers (Amika Corp.), which contains two 75-μl chambers separated by a 5-kDa cut-off dialysis membrane. Equilibration conditions were 50 mm Tris, 50 mm Bis-Tris, 100 mm sodium acetate, pH 7.5, and 32.5 μm yGCN5. The K d value for AcCoA in the presence of yGCN5 was determined by transferring 10–400 μm AcCoA (20–40 cpm 3H/pmol) into the buffer chamber and yGCN5 into the sample chamber. After 48 h of equilibration on a level shaker, samples were recovered from each chamber and counted by liquid scintillation to determine the amount of radioactivity in the buffer chamber ([AcCoA]free) and the sample chamber ([AcCoA]free + [AcCoA·yGCN5]). The calculated [AcCoA]free was subsequently subtracted from the calculated concentration [AcCoA]free + [AcCoA·yGCN5] to determine the concentration of bound AcCoA. The data were presented in hyperbolic form (Equation 7). [AcCoA·yGCN5]=([yGCN5] tot·[AcCoA] free)/(Kd+[AcCoA] free)Equation 7 εCoA was titrated with increasing [yGCN5]. [εCoA] was determined spectrophotometrically by using a molar extinction coefficient of 5.6 mm−1cm−1 at 275 nm. The anisotropy measurements were performed on a Photon Technologies QM-1 steady-state fluorescence spectrophotometer equipped with a thermostat. εCoA was excited at 305 nm (3-nm slits), and emission was monitored at 405 nm with 20-nm slits for both the parallel and perpendicular components. Measurements were performed in 50 mm Tris, pH 7.5, 25 °C, using a 200-μl quartz cuvette. A concentrated solution of yGCN5 (in 1-μl increments) was added to the cuvette containing 0.5 μm εCoA and mixed using a small magnetic stirring bar. Anisotropy increases were measured after 1–2 min. The anisotropy r of εCoA was calculated from the parallel Ivv and perpendicularIvh polarized fluorescence intensities measured upon parallel excitation according to Equation 8, r=(Ivv−G·Ivh)/(Ivv+2·G·Ivh)Equation 8 where G is the ratio of the parallel Ivhand perpendicular Ivh polarized fluorescence intensity measured upon parallel excitation to correct for the different efficiencies of the parallel and perpendicular polarization fluorescence detectors. Data were fitted to Equation 9 described in Richards et al. (22.Richards J.P. Bachinger H.P. Goodman R.H. Brennan R.G. J. Biol. Chem. 1996; 271: 13716-13723Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), A=((Abound−Afree)·[yGCN5]/Kd+[yGCN5])+AfreeEquation 9 where A is the anisotropy measured at a given concentration of yGCN5, and A free andA bound are the anisotropies of free and bound εCoA, respectively. This simplified equation can be used when the dissociation constant is ≥10-fold higher than the fluorescent ligand concentration, since the concentration of yGCN5 bound to εCoA becomes negligible in comparison to the total yGCN5 concentration (22.Richards J.P. Bachinger H.P. Goodman R.H. Brennan R.G. J. Biol. Chem. 1996; 271: 13716-13723Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Given the highly charged characteristic of the nucleosomal complex, it is likely that ionic strength will greatly influence the catalytic activity of these enzymes. Therefore, the effect of ionic strength on the yGCN5 HAT reaction was examined by varying NaCl concentrations between 0 and 1 m, and the resulting HAT activity was monitored in the radioactive P81-filter binding assay as described under “Experimental Procedures.” The resulting yGCN5 activity was plotted versus the log of the ionic strength and fitted to a line using least squares analysis (Fig.1). A direct linear relationship between the log of the ionic strength and inactivation of yGCN5 HAT activity was observed. There was an approximate 7-fold decrease of yGCN5 HAT activity between 0.15 m NaCl and 1 m NaCl. Yeast GCN5 catalyzes the transfer of an acetyl moiety from AcCoA to the ε-amino side chain of Lys 14 of histone H3 (Scheme 1). It was first necessary to determine the basic kinetic mechanism (sequential or ping-pong) utilized by yGCN5 before a detailed investigation into the order of substrate binding and the order of product release could be performed. CoA-dependent transferases are known to utilize one of two distinct mechanisms to catalyze acetyl group transfer. One mechanism involves acetyl transfer from CoA to an enzyme side chain nucleophile before transfer to the amine substrate (23.Thompson S. Mayerl F. Peoples O.P. Masamune S. Sinskey A.J. Walsh C.T. Biochemistry. 1989; 28: 5735-5742Crossref PubMed Scopus (84) Google Scholar). In a subsequent step, the enzyme then transfers this acetyl group to the acceptor amine substrate. The alternative mechanism involves direct acetyl transfer from AcCoA to the amine substrate acceptor without the formation of a covalent enzyme intermediate (24.Lewendon A. Murray I.A. Shaw W.V. Gibbs M.R. Leslie A.G. Biochemistry. 1994; 33: 1944-1950Crossref PubMed Scopus (62) Google Scholar). The latter mechanism requires that both substrates and enzyme must form a ternary complex before catalysis can occur. In a previous study (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) we performed a Bi-substrate kinetic analysis with core histone proteins (H3, H2A, H2B, and H4) derived from calf thymus and demonstrated that yGCN5 utilizes a sequential (ternary complex) mechanism. Here, a synthetic H3 peptide (ARTKQTARKSTGGKAPPKQLC) was employed as a substrate in lieu of calf thymus histones since this substrate provides a better defined system to analyze the steady-state kinetic parameters. To distinguish between a ternary complex (sequential) mechanism and a ping-pong (covalent-intermediate) mechanism, initial velocity steady-state kinetic parameters were obtained for AcCoA and H3 peptide using AcCoA concentrations spanning 1–20 μm and H3 peptide concentrations spanning 25–730 μm. Analysis of these data by plotting 1/velocity against 1/[H3 peptide] at different fixed concentrations of AcCoA resulted in an intersecting line pattern (Fig. 2) that is characteristic of a ternary complex (sequential) mechanism. In contrast, a ping-pong (covalent intermediate) mechanism is typically characterized by a parallel line pattern. The identification of a ternary complex was consistent with our previously published report (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) using a histone preparation derived from calf thymus, thus validating the use of H3 peptide as an excellent analog of histone protein. The basic steady-state parameters derived from this Bi-substrate analysis are summarized in Table I. Employing acetylated (Lys14Ac) synthetic H3 peptide as a substrate for yGCN5, it was determined that lysine 14 of H3 peptide is the only residue of H3 peptide acetylated. No other Lysine (Lys-4, Lys-9, or Lys-18) of H3 peptide was found to be acetylated above background levels when using either the radioactive P81-filter binding assay or the coupled enzyme spectrophotometric assay (data not shown). This is an important result since acetylation at a secondary site to lysine 14 of H3 peptide would significantly complicate the initial velocity kinetic analysis.Table ISummary of kinetic and equilibrium constants for yeast GCN5 HATSubstratesK mk cat/K mk catAcCoA2.5 ± 1.4 μm aExperiments were performed in triplicate. The reported value is an average ± S.D.6.8 × 105 m−1 s−11.7 ± 0.12 s−1 aExperiments were performed in triplicate. The reported value is an average ± S.D.H3 peptide0.49 ± 0.08 mm aExperiments were performed in triplicate. The reported value is an average ± S.D.3.5 × 103 m−1 s−1InhibitorsK iiK isK dCoA6.7 ± 5.1 μm aExperiments were performed in triplicate. The reported value is an average ± S.D.Desulfo-CoA33 ± 1.3 μm aExperiments were performed in triplicate. The reported value is an average ± S.D.Etheno-CoA5.1 ± 1.1 μmAcH3 peptide (vs. CoA)2.2 ± 0.40 mm aExperiments were performed in triplicate. The reported value is an average ± S.D.1.9 ± 0.15 mm aExperiments were performed in triplicate. The reported value is an average ± S.D.AcH3 peptide (vs. H3)2.5 ± 1.3 mm aExperiments were performed in triplicate. The reported value is an average ± S.D.2.1 ± 0.90 mm aExperiments were performed in triplicate. The reported value is an average ± S.D.a Experiments were performed in triplicate. The reported value is an average ± S.D. Open table in a new tab Several distinct kinetic mechanisms are possible for enzyme systems that employ two substrates and produce two products. Our previous study (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. Denu J.M. J. Biol. Chem. 1999; 274: 18157-18160Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) combined with the Bi-substrate analysis described above have strongly argued against a ping-pong (covalent intermediate) type mechanism and demonstrated that yGCN5 followed a sequential Bi-Bi mechanism. The binding of substrates and release of products can be random, fully ordered, or a combination of both. Product inhibition studies can easily distinguish among the various possibilities. To distinguish among the various possible kinetic models for substrate binding and product release, the products of the yGCN5-catalyzed HAT reaction were used to inhibit the yGCN5-catalyzed HAT reaction. To correctly analyze product inhibition data, the reversibility of the yGCN5-catalyzed reaction was examined. This was accomplished by a high performance liquid chromatography assay using reversed phase chromatography to directly monitor the formation of deacetylated H3 peptide from a reaction of yGCN5, CoA, and acetylated (Lys14Ac) H3 peptide. Analysis of the time points from a reaction carried out at pH 7.5 and 25 °C with high concentrations of acetylated (Lys14Ac) H3 peptide and CoA with a corresponding non-enzyme control were subjected to reversed phase chromatography. The estimated turnover number under this set of conditions (1.0 mm CoA, 1.0 mm acetylated (Lys14Ac) H3 peptide) was 0.00002 s−1, ∼100,000-fold lower than thek cat value (1.7 s−1) determined for the forward reaction under saturating substrates. Thus, yGCN5 is capable of being a histone deacetylase as is predicted from thermodynamics, albeit much less efficiently. Products of the yGCN5-catalyzed HAT reaction are classified as competitive inhibitors if increasing concentrations of product decrease the apparent k cat/K m for the varied substrate, uncompetitive if they decrease the apparentk cat, and noncompetitive if they decrease both. Evaluation of the patterns are obtained by observing increases in the slopes (apparent K m/k cat), the intercepts (apparent 1/k cat), or both, respectively, for lines in double-reciprocal plots of 1/v versus 1/[S] obtained at increasing concentrations of product (and a constant concentration of the second substrate). CoA was evaluated as a product inhibitor against both AcCoA and H3 peptide. The experiment was performed using the radioactive P81-filter binding assay since CoA itself is a substrate for the coupled enzyme in the spectrophotometric assay (19.Kim Y. Tanner K.G. Denu J.M. Anal. Biochem. 2000; 280: 308-314Crossref PubMed Scopus (83) Google Scholar). Initial velocities were determined, and the data were plotted in double-reciprocal form with 1/velocity versus1/[AcCoA] at several fixed concentrations of CoA. A series of double-reciprocal straight line plots intersected on the 1/velocity ordinate, indicating a competitive inhibition mechanism. This indicates that at high concentrations of AcCoA, the inhibition by CoA is overcome, and the V max values approach identical levels. CoA was found to be a linear competitive inhibitor (Fig.3) versus AcCoA at 100 μm H3 peptide, with a K is of 6.7 ± 5.1 μm, similar to the value obtained for (Fig. 2) theK m for AcCoA (4.5 ± 3.6 μm). The replot of the slopes from the double-reciprocal plot as a function of [CoA] was linear and yielded similar inhibition constants (analysis not shown). These data indicated that AcCoA and CoA compete for the same form of yGCN5 and the same mutually exclusive binding site. In a sequential mechanism involving two substrates and two products, this requires that AcCoA and CoA bind to free enzyme; thus, AcCoA is the first substrate to add, and CoA is the last product to leave. In a complimentary experiment, CoA did not inhibit against H3 peptide when AcCoA was present at saturating levels (data not shown). These results are consistent with CoA and H3 peptide binding to different forms of yGCN5 and are consistent with the kinetic mechanism in which AcCoA binds first, followed by binding of H3 peptide and subsequent transfer of the acetyl group from AcCoA to H3 and an ordered release of products with acetylated (Lys14Ac) H3 peptide releasing before the release of CoA (Scheme FS2).Figure FS2Proposed kinetic mechanism of yGCN5.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further demonstrate that H3 peptide and core histones operate through the same kinetic mechanism, CoA inhibition studies were carried out using calf thymus core histones as the amine nucleophile substrate (Fig. 4). As with H3 peptide (Fig. 3), CoA is a pure competitive inhibitor against AcCoA when calf thymus core histones are used. Again, this indicated that AcCoA is the first substrate to add, and CoA is the last product to be released. By default, H3 peptide binds second and acetylated (Lys14Ac) H3 peptide is the first to leave. The inhibition constants for CoA are summarized in Table I. Taken together with the fact that k catvalues are similar between the two sources of substrate (k cat(H3) = 1.7 s−1,k cat(histones) = 0.74 s−1) and that both substrates (H3 peptide and calf thymus histones) generate intersecting line patterns in Bi-substrate kinetics, this strongly suggests that H3 peptide and core histones follow the same kinetic mechanism. To further establish that CoA acts as a pure competitive inhibitor, we utilized the CoA derivative desulfo-CoA in steady-state inhibition studies. Desulfo-CoA (which lacks only the terminal sulfhydryl of CoA) is a dead-end inhibitor since it forms a non-productive complex with yGCN5 and H3 peptide. Initial velocities were obtained, and the data were plotted in double-reciprocal form with 1/velocity versus 1/[AcCoA] at several fixed concentrations of desulfo-CoA. Desulfo-CoA was found to be a linear competitive inhibitor" @default.
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• W1963753695 date "2000-07-01" @default.
• W1963753695 modified "2023-10-18" @default.
• W1963753695 title "Kinetic Mechanism of the Histone Acetyltransferase GCN5 from Yeast" @default.
• W1963753695 cites W1511815964 @default.
• W1963753695 cites W1641913445 @default.
• W1963753695 cites W1646199134 @default.
• W1963753695 cites W1763859619 @default.
• W1963753695 cites W1825285599 @default.
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• W1963753695 cites W1968058162 @default.
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