期刊
BIOCHEMISTRY
卷 39, 期 39, 页码 11961-11969出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi001272h
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资金
- NIDDK NIH HHS [T32 DK07680] Funding Source: Medline
- NIGMS NIH HHS [GM 59785-01] Funding Source: Medline
Human transcriptional coactivator P/CAF (p300/CBP-associating factor) is a histone acetyltransferase (HAT) and is a member of the GNAT (GCN5 related N-acetyltransferases) superfamily. P/CAF was originally identified by its ability to activate transcription of a variety of genes through its interaction with p300/CBP, Though Lys-14 of histone H3 appears to be the preferred substrate, other nonhistone proteins can also serve as substrates for P/CAF. However, few studies have addressed the catalytic and kinetic mechanisms of histone/protein acetylation by P/CAF. In this study, we have systematically determined the kinetic mechanism for P/CAF, identified the critical ionizations for binding/catalysis, and established the rate-limiting step in turnover. This was accomplished by a variety of approaches including FH-dependent activity measurements, Bi-substrate kinetic analysis, authentic product inhibition by coenzyme A (CoA) and acetylated H3 (Ac-Lys-14) peptide, direct measurements of substrate/product binding affinities (equilibrium dialysis), and a pre-steady-state quench-flow analysis. The results are consistent with a fully ordered Bi-Bi kinetic mechanism, where chemical catalysis is rate-determining Acetyl-CoA (AcCoA) binds with high affinity (K-d = 0.64 +/- 0.12 mu M) to the free form of the enzyme. Histone H3 peptide binds (apparent K-d = 116 +/- 17 mu M) only after AcCoA is bound. No H3 peptide binding to the free enzyme was detectable, In the ternary complex, the epsilon-amino of Lys-14 (H3 peptide substrate) directly attacks the carbonyl carbon of AcCoA, transferring the acetyl group to the acceptor peptide substrate (rate-limiting step). Products are released in an ordered fashion, with Ac-Lys-ld, H3 released first followed by release of CoA, The pH dependency of the k(cat)/K-m parameter revealed two P/CAF ionizable groups (pK(a) values of 6.9 and 7.5) that must be unprotonated for activity. The group with a pK(a) value 7.5 was assigned to Glu-570, which is the proposed general base catalyst, abstracting a proton from the epsilon-amino group and facilitating nucleophilic attack.
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