A new set of experimental kinetic data on the hydrolysis of a series of phenylacetylp-substituted anilides catalyzed by penicillin G acylase from Escherichia coli(PGA) is presented in this article. The Hammett plot of log(kcat,R ⁄kcat,H) versus rp ) has three linear segments, which distinguishes the enzyme from the other N-terminal nucleophile hydrolases for which data are available.