Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined 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 strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the 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 by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (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) × 10-3 s-1 for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate 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 values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D2O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [kHinact/ kDinact = 0.65 ± 0.02 and (k2/K macetyl)H/(k2/Km acetyl)D = 0.60 ± 0.03], which were found to be identical to the fractionation factors (Φ) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S--His-ImH +). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible 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 to rapidly form a stable acetylated species poised to react with an arylamine substrate.