Probing the Mechanism of Hamster Arylamine N-Acetyltransferase 2 Acetylation by Active Site Modification, Site-Directed Mutagenesis, and Pre-Steady State and Steady State Kinetic Studies
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- 作者：Haiqing Wang ; Gregory M. Vath ; Kara J. Gleason ; Patrick E. Hanna ; and Carston R. Wagner
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 29, 2004
- 年：2004
- 卷：43
- 期：25
- 页码：8234 - 8246
- 全文大小：264K
- 年卷期：v.43,no.25(June 29, 2004)
- ISSN：1520-4995
文摘
Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzymeA (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recentlydetermined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictlyconserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatmentof NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivatedthe enzyme with second-order rate constants of 802.7 ± 4.0 and 426.9 ± 21.0 M-1 s-1, respectively.MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation byIAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoaceticacid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenylacetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 ± 0.05) × 105 M-1 s-1, followed by a slow linear rate of (7.85 ± 0.65) × 10-3s-1 for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and therate of acetylation with PNPA revealed similar pKa1 values of 5.23 ± 0.09 and 5.16 ± 0.04, respectively,and pKa2 values of 6.95 ± 0.27 and 6.79 ± 0.25, respectively. Both rates reached their maximum valuesat pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D2O revealed a largeinverse solvent isotope effect on both inactivation and acetylation of NAT2 [kHinact/kDinact = 0.65 ± 0.02and (k2/Kmacetyl)H/(k2/Kmacetyl)D = 0.60 ± 0.03], which were found to be identical to the fractionation factors(
) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of thecatalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structuralintegrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism ofNAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S--His-ImH+). However, incontrast to the case with cysteine proteases, a pH-dependent protein conformational change is likelyresponsible for the second pKa, and not deprotonation of the thiolate-imidazolium ion. In addition,substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered torapidly form a stable acetylated species poised to react with an arylamine substrate.
) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of thecatalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structuralintegrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism ofNAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S--His-ImH+). However, incontrast to the case with cysteine proteases, a pH-dependent protein conformational change is likelyresponsible for the second pKa, and not deprotonation of the thiolate-imidazolium ion. In addition,substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered torapidly form a stable acetylated species poised to react with an arylamine substrate.