Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?
A new set of experimental kinetic data on the hydrolysis of a series of phenylacetyl p-substituted anilides catalyzed by penicillin G acylase from Escherichia coli (PGA) is presented in this article. The Hammett plot of log(k(cat,R)/k(cat,H)) versus sigma(p) (-) has three linear segments, which distinguishes the enzyme from the other N-terminal nucleophile hydrolases for which data are available. Three amino acids in the vicinity of the catalytic SerB1 (AsnB241, AlaB69, and GlnB23) were included in the quantum mechanical model. The stable structures and the transition states for acylation were optimized by molecular mechanical modeling and at the AM1 level of theory for three model substrates (with H, a methoxy group or a nitro group in the para position in the leaving group). Intrinsic interactions of several functional groups at the active site of PGA are discussed in relation to the catalytic efficiency of the enzyme. The energy barrier computed for the first step of acylation (the
