Mechanistic studies on metallohydrolases and metallohydratases

Methionine aminopeptidases (MetAPs) are metalloenzymes responsible for the hydrolytic cleavage of the initiator N-terminal methionine residue from newly synthesized polypeptide chains in living organisms. On the other hand, nitrile hydratases (NHases) are metalloenzymes that catalyze the hydration of nitriles to the corresponding higher commercial value amides under mild conditions. The primary goals of this research were to understand the catalytic mechanism of peptide hydrolysis by MetAP, and other enzymes belonging to the "pita bread" superfamily, as well as the reaction mechanism of nitrile hydration by NHase.;Crystallographically, MetAPs have a dinuclear active site. However, in early 2000, it was suggested that MetAPs could function as mononuclear enzymes. This obscured the role of the second metal ion and the function of an aspartate residue, D97, that is a bidentate ligand to the second metal ion. This aspartate residue was studied in detail by generating variants with altered aspartate residue. The data suggested that the presence of aspartate at the 97 position is critical and alterations at the D97 position affect the binding properties of the catalytically important first metal ion.;A direct and convenient assay for MetAP was developed and was utilized to screen novel inhibitors. During the course of the research work, it was found that a distant residue affected the catalysis of type-I MetAP. This residue was found to affect the catalysis of smaller substrates. Owing to the importance of MetAP inhibitors, the binding of two different kinds of potential MetAP inhibitors to both type-I and -II MetAPs was studied in great detail.;Even though a significant amount of structural information is now available for NHases, details of the enzymatic reaction remain unknown. The research presented herein focuses on the purification and detailed kinetic characterization of a thermally stable Co-type NHase. Based on these data, a novel catalytic mechanism is proposed for NHase enzymes.