Kinetic Mechanism of the Histone Acetyltransferase GCN5 from Yeast

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. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) of yGCN5 the conserved as a in the GCN5 HAT the ε-amino group of lysine 14 in histone the of HAT a for the between histone acetylation and In study have determined the overall kinetic mechanism, developed methods for binding affinities and equilibrium and determined the of binding and the of product by the product CoA and (Lys14Ac) H3 peptide and the dead-end inhibitor The results are with a kinetic mechanism, where AcCoA is the to and CoA is the last product were of the H3 and the H3 peptide to the amino-terminal residues of histone H3 and an were by the The of was from histones were from was from were from were from 1-N 6-etheno-CoA was from were from The catalytic of yGCN5 was by for and from as (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. K.G. Biochem. PubMed Scopus Google Scholar). on by were and and to on was by and were and were determined by the of M.M. Biochem. PubMed Scopus Google Scholar). yGCN5 histone acetyltransferase was using a as Y. K.G. Biochem. PubMed Scopus Google Scholar). the in the HAT was by using a with The of was by the of to which was The HAT 1 and of of is by the to the of of to The was and and were and were by the of The were for to and from the of CoA were from the from the these the the Y. K.G. Biochem. PubMed Scopus Google Scholar). Also, the of the kinetic were to the These were typically for to of the H3 peptide (Lys14Ac) H3 peptide were in the of yGCN5 was using the binding as (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The Bi-substrate kinetic analysis was AcCoA and H3 peptide histone The yGCN5 HAT was the using The data were to kinetic the sequential the and the using the of Mol. Biol. Google Scholar) and the and a 1 The product CoA and (Lys14Ac) H3 as as the analog the of were in steady-state inhibition To competitive noncompetitive inhibition was the data were to the inhibition on the by Mol. Biol. Google Scholar) using a was using which two by a dialysis were and The K d value for AcCoA in the of yGCN5 was determined by AcCoA the and yGCN5 the of on a were from and by to the of in the and the The was from the to the of AcCoA. The data were in form εCoA was with was determined by using a of The were on a steady-state fluorescence with a εCoA was and was with for both the and were in using a A of yGCN5 was to the εCoA and using a magnetic were The of εCoA was from the and fluorescence to 8 where is the of the fluorescence to for the of the and fluorescence were to 9 in R.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), 9 where A is the a of yGCN5, and A are the of and This the is than the fluorescent the of yGCN5 to εCoA in to the yGCN5 R.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the highly of the nucleosomal is that the catalytic of these the of on the yGCN5 HAT was by between and 1 and the HAT was in the binding as The yGCN5 was versus the of the and to a using analysis A between the of the and of yGCN5 HAT was was an of yGCN5 HAT between and 1 GCN5 catalyzes the transfer of an acetyl moiety from AcCoA to the ε-amino side of 14 of histone H3 (Scheme 1). was necessary to the kinetic by yGCN5 before a detailed the of binding and the of product performed. are to of two distinct mechanisms to acetyl group acetyl transfer from CoA to an side before transfer to the S. F. S. A.J. Biochemistry. PubMed Scopus Google Scholar). In a the acetyl group to the The acetyl transfer from AcCoA to the the of a A. Biochemistry. PubMed Scopus Google Scholar). The that both substrates and must form a ternary complex before In a study (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) a Bi-substrate kinetic analysis with core histone H2B, and from and demonstrated that yGCN5 a sequential a synthetic H3 peptide was as a in of histones a to the steady-state kinetic To between a ternary complex and a mechanism, steady-state kinetic were obtained for AcCoA and H3 peptide using AcCoA and H3 peptide of these data by against of AcCoA in an that is of a ternary complex In a is typically by a The of a ternary complex was with (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) using a histone from the of H3 peptide as an analog of histone The steady-state from Bi-substrate analysis are in Employing (Lys14Ac) synthetic H3 peptide as a for yGCN5, was determined that lysine 14 of H3 peptide is the of H3 peptide of H3 peptide was found to levels using the binding the This is an acetylation a site to lysine 14 of H3 peptide the kinetic of kinetic and equilibrium for yeast GCN5 ± were in The reported value is an ± ± were in The reported value is an ± ± were in The reported value is an ± ± 5.1 were in The reported value is an ± ± were in The reported value is an ± ± 1.1 peptide ± were in The reported value is an ± ± were in The reported value is an ± peptide ± were in The reported value is an ± ± were in The reported value is an ± were in The reported value is an ± in a Several distinct kinetic mechanisms are for that two substrates and two study (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) with the Bi-substrate analysis have against a and demonstrated that yGCN5 followed a sequential The binding of substrates and of a of Product inhibition studies the various To the various kinetic models for binding and product the of the HAT were to the HAT To product inhibition the of the was This was by a high using to the of H3 peptide from a of yGCN5, and (Lys14Ac) H3 of the from a and with high of (Lys14Ac) H3 peptide and CoA with a were to The of (Lys14Ac) H3 was than value determined for the yGCN5 is of a histone as is from albeit much of the HAT are as competitive of product the for the the and noncompetitive of the are obtained by in the K the for in of versus obtained of product a of the CoA was as a product inhibitor against both AcCoA and H3 The was using the binding CoA is a for the in the Y. K.G. Biochem. PubMed Scopus Google Scholar). were and the data were in form with several of A of on the a competitive inhibition This that high of the inhibition by CoA is and the CoA was found to a competitive inhibitor versus AcCoA H3 with a K is of 6.7 ± 5.1 similar to the value obtained for theK for AcCoA ± The of the from the as a of was and similar inhibition These data indicated that AcCoA and CoA for the form of yGCN5 and the binding In a sequential two substrates and two that AcCoA and CoA bind to AcCoA is the to and CoA is the last product to leave. In a CoA against H3 peptide AcCoA was levels These results are with CoA and H3 peptide binding to of yGCN5 and are with the kinetic in which AcCoA binds first, followed by binding of H3 peptide and transfer of the acetyl group from AcCoA to H3 and an of with (Lys14Ac) H3 peptide before the of CoA (Scheme kinetic of To that H3 peptide and core histones the kinetic mechanism, CoA inhibition studies were using core histones as the with H3 peptide CoA is a competitive inhibitor against AcCoA core histones are indicated that AcCoA is the to and CoA is the last product to H3 peptide binds and (Lys14Ac) H3 peptide is the to leave. The inhibition for CoA are in with the that are similar between the two of and that both substrates peptide and in Bi-substrate suggests that H3 peptide and core histones the kinetic To that CoA as a competitive inhibitor, the CoA in steady-state inhibition the of is a dead-end inhibitor a complex with yGCN5 and H3 were and the data were in form with versus several of was found to a competitive inhibitor versus AcCoA histone H3 with a K is of ± The binding for CoA and were with K of 6.7 and The of the in must for the K is value for with The of the results in a of with residues in the CoA binding site (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). Together, these data that CoA bind to the form of the and are with the kinetic of equilibrium the for AcCoA binding to yGCN5 was of were the and yGCN5 was the were from and were by The data were as and the A binding is shown in The value from a K d value of 8.5 ± 2.6 and an of ± These data are in with the determined by the of M.M. Biochem. PubMed Scopus Google and the K i value obtained from kinetic methods 6.7 in fluorescence are to in the fluorescent of yGCN5 binding AcCoA. an fluorescent fluorescence J.R. M. Mol. 1996; PubMed Scopus (216) Google Scholar). fluorescence for εCoA binding to yGCN5 were to the εCoA a fluorescent of two that has been to study and enzymes 1997; PubMed Scopus Google Scholar, B.M. D. Biochem. 1996; PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar, B.M. J. Sci. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar). structures of CoA and GCN5 demonstrated that are between GCN5 and the moiety of CoA (15.Clements A. Rojas J.R. 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Nature. 1999; 401: 93-98Crossref PubMed Scopus (234) Google Scholar). that εCoA as a CoA binding yGCN5, εCoA a in fluorescence in to a binding and to the a of 5.1 ± 1.1 A in which AcCoA was in the of the in fluorescence The K d value is in with the K is of 6.7 obtained for CoA that εCoA is a for CoA in these binding To the product inhibition the product inhibitor (Lys14Ac) H3 peptide was as an both H3 peptide and AcCoA. by the kinetic (Scheme both were found to noncompetitive that the bind to and that is a between the two of binding the were that the is that the is than for the a for the The steady-state kinetic inhibition for (Lys14Ac) H3 peptide are in These in with the CoA and inhibition are with yGCN5 HAT kinetic The kinetic of two by both substrates John Scholar). is that a analysis that an suggests a ternary complex mechanism, and a is of a This has been to the of acetyltransferases S. F. S. A.J. Biochemistry. PubMed Scopus Google Scholar, A. Biochemistry. PubMed Scopus Google Scholar). the Bi-substrate with yGCN5 is with a ternary complex These data are with a of lysine on AcCoA. is to the of a by has been for an were to a yGCN5 was with AcCoA. G. M. R. Y. and J. M. an is in the yGCN5 must both AcCoA and H3 peptide are and must before H3 peptide CoA is data suggest that yGCN5 a ternary complex with AcCoA and H3 peptide before catalysis. In a synthetic peptide to the amino-terminal amino of histone was demonstrated that of histones also an from similar Bi-substrate kinetic (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the obtained using are Together, these results that both the H3 peptide and H3 the core the kinetic and that synthetic H3 peptide is a and in detailed In the synthetic peptide a of histones from the synthetic H3 peptide is The acetylation of histones from is and the acetylation is with H3 peptide are lysine acetylation with which core histones H3, H2B, with are to the synthetic (Lys14Ac) H3 as a product inhibitor for the HAT These have to the kinetic mechanism, as in The solved structures and of GCN5 and GCN5 have the of a ternary complex of and H3 peptide (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). The molecular models revealed two that are to of the structures have AcCoA CoA to the of the revealed a ternary complex of CoA to the and an H3 to the The that the acetyl moiety of AcCoA was to the in the binary complex ofTetrahymena is with the of a catalysis. of the ternary complex revealed that is for both H3 peptide and AcCoA to This dead-end complex similar to that of a ternary complex of GCN5 with both substrates AcCoA and H3 the of the the group and of The structure that is to the acetyl moiety of AcCoA. In the of the acetyl group to both the ε-amino group of lysine 14 and the proposed which must the ε-amino group of lysine 14 before on the of AcCoA. demonstrated that the conserved was as a (18.Tanner K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). This study revealed an of a that must for a of ± and the was to that was as a the ε-amino group of lysine 14 from histone was that two large the of This is with the a 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 K.G. Trievel R.C. Kuo M.H. Howard R.M. Berger S.L. Allis C.D. Marmorstein R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The Bi-substrate kinetic analysis suggests that a ternary complex is by GCN5. To the of binding and the of product product inhibition studies were The of product inhibition is for a kinetic John Scholar). and product are levels of the and the the of The two HAT (Lys14Ac) H3 peptide and were as product The of was from Acetylated (Lys14Ac) H3 peptide was as an inhibitor versus both H3 peptide and AcCoA by the kinetic (Scheme both were found to noncompetitive that the bind to and that is a between the two of Acetylated (Lys14Ac) H3 peptide demonstrated noncompetitive inhibition with to that (Lys14Ac) H3 peptide and AcCoA bind to the form of yGCN5 AcCoA binds to H3 peptide binds to Also, (Lys14Ac) H3 peptide noncompetitive inhibition with to H3 that and H3 peptide bind to H3 peptide binds to H3 peptide binds to the were that the is that the is a for the The product CoA competitive inhibition AcCoA that AcCoA and CoA for the In a sequential mechanism, that acetyl-CoA and CoA bind to AcCoA is the to and CoA is the last product to released by the kinetic CoA against H3 AcCoA is levels with CoA as a competitive inhibitor, the CoA in steady-state inhibition studies and found that also as a competitive inhibitor with to acetyl-CoA is a dead-end inhibitor a with the that (Lys14Ac) H3 peptide is a noncompetitive inhibitor against both H3 peptide and AcCoA suggests that AcCoA is the to bind yGCN5 followed by H3 transfer of the acetyl moiety from AcCoA to H3 peptide in the ternary (Lys14Ac) H3 peptide must first, followed by the of CoA (Scheme The data suggest that H3 peptide is of binding to yGCN5, kinetic and biochemical that highly conserved family of GCN5 HATs a kinetic mechanism, is that binding of AcCoA to yGCN5 a that the for H3 peptide with a solved structure of a family member, with a Bi-substrate that binding of AcCoA a that the binding site for the F. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This is in with the kinetic of proposed by and J. J. P.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). have demonstrated for yGCN5, was shown to an the structure of peptide ternary complex suggests that the binding of H3 peptide is on AcCoA binding 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). The binding of AcCoA was to the histone binding the binding affinities of substrates and is an the catalytic and kinetic mechanisms of the HAT developed a steady-state fluorescence for εCoA binding to εCoA 6-etheno-CoA), a fluorescent of is to CoA for two within the The structures of the binary and ternary of GCN5 with CoA AcCoA that a the of the contacts the group and of In the in these structures is highly with specific with GCN5 residues (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). The binding from these a of 5.1 This value is in with theK is of 6.7 for CoA determined in the inhibition equilibrium a of 8.5 ± 2.6 was determined for AcCoA binding to The that the determined by these binding were in suggests that AcCoA and CoA bind with similar binding affinities to The that the binding affinities between AcCoA and product CoA are similar that the of AcCoA/CoA a for in vivo HAT This for HATs transcriptional with various that the of to is a of HAT