Catalytic mechanism of the hamster arylamine N-acetyltransferase 2 (NAT2) and human histidine triad nucleotide binding protein 1 (HINT1)

By covalent catalysis, enzymes utilize the key residue to form an intermediate with the reactant, leading to an entropically favored unimolecular reaction over a bimolecular reaction. Here we studied two enzymes that employ covalent catalysis, hamster N-acetyltransferase 2 (NAT2) and human histidine triad nucleotide binding protein 1 (HINT1). Our results with hamster NAT2 demonstrated how an enzyme's catalysis can be modulated by small changes in the active site and how these changes affect the subsequent intracellular fate of the enzyme. Also, our studies on human Hint1 provided mechanistic details of the hydrolysis of phosphoramidates and acyl adenylates, elucidated the specific roles of important residues, and shed light on the HIT superfamily in general.;Arylamine N-acetyltransferase is an important phase II drug metabolizing enzyme that contains a catalytic triad of Cysteine-Histidine-Aspartate. It catalyzes the transfer of an acetyl group from acetyl-coenzyme A to arylamine subsrates by formation of an acetylated enzyme intermediate at the active site cysteine. The proximal conserved tyrosine 190 in hamster NAT2 forms a hydrogen bond with one of the triad residues, Asp122, and a potential pi-pi interaction with the active site His107. Site-directed mutagenesis, transient state and steady state kinetics were applied, as well as the dependence of the reaction rate on the pH and substrate basicity, to reveal that this tyrosine is critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of arylamines, as well as affecting the pKa of the active site cysteine and histidine. However, structural analysis indicated these effects are through the alteration of the local conformation of the active site rather than the overall folding. Furthermore, although the tyrosine 190 mutants have shortened half life of the acetylated NAT intermediate, their intracellular stability and protein degradation are not affected, in contrast to R64W and C68Y mutations which caused hamster NAT2 to be constitutively ubiquitylated and formed microaggregates in cultured cells. This result suggested that the acetylation state of NAT may not be determinant of the protein half life in vivo.;Human HINT1 is a homodimeric protein featuring an active site motif of His-X-His-X-His-XX, where X represents a hydrophobic amino acid. It catalyzes the hydrolysis of phosphoramidates and acyl adenylates with a high efficiency. The reaction mechanism is consistent with a double displacement mechanism, in which the active site nucleophile Histidine 112 is first adenylated by the substrate, followed by hydrolysis of the AMP-Hint1 intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicates that the enzyme adenylation is faster than the subsequent hydrolysis of intermediate and product release, and that the two active sites function independently. Solvent viscosity experiments and catalytic trapping experiments suggest that both chemical transformation and product dissociation limit the overall turnover, and specifically, the off rate of final product AMP is at least limiting. To further understand the nature of groups that are critical for catalysis and/or binding, pH profiles, solvent isotope, and proton inventory studies were carried out. The Hint1 adenylation rate is found to be dependent on two residues with pKas of 6.5 and 8, and the former pKa agrees well with the NMR titration results for the pKa of the active site nucleophile His112. Proton inventory and pH dependence studies of Hint adenylation revealed that the active site His114 is likely to act as the general acid/base and be responsible for one proton transfer during the enzyme adenylation. Taken together, the data are consistent with a mechanism that relies on a fast adenylation step while being partially rate limited by an internal step (hydrolysis) and an external step (product release).