| Escherichia coli leader peptidase I (leader peptidase) is essential for the viability of the bacterium and it is thus a potential anti-bacterial drug target. Previous studies suggested that leader peptidase is a novel serine protease utilizing a Ser-Lys catalytic dyad. In this work I describe the development of a continuous assay for leader peptidase activity and a series of biochemical studies to elucidate its catalytic mechanism.;Based on the fluorescence resonance energy transfer principle, an internally quenched fluorescent peptide, Y(NO;In an effort to understand further the role of Lys as a general base in the catalysis, pH dependence studies of leader peptidase activity were performed and an ionizable group with pKa value of 6.7 was observed in the free enzyme. To assign this basic residue, Lys-145 was mutated to Arg by site-directed mutagenesis yielding an inactive enzyme at all pHs (7.0 to 10.0). These observations favor Lys-145 as an important residue in the leader peptidase catalyzed reaction. Furthermore, an acyl-enzyme intermediate was observed employing a product partitioning approach using hydroxylamine as a nucleophile. The initial velocity of the reaction displayed a linear dependence on hydroxylamine concentration, indicating that the hydrolysis of the acyl-enzyme intermediate is the rate-limiting step in the overall reaction. |
