MUTATIONAL ANALYSIS OF THE ACTIVE-SITE OF PSEUDOMONAS-FLUORESCENS PYRROLIDONE CARBOXYL PEPTIDASE

On the basis of chemical inhibition studies and a multiple alignment o f four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seve n conserved residues of the Pseudomonas fluorescens Pcp, which might b e important for enzyme activity, have been modified by site-directed m utagenesis experiments, Wild-type and mutant Pcps were expressed in Es cherichia coli, purified, and characterized by the ability to cleave t he synthetic chromogenic substrate pyroglutamyl-beta-naphthylamide and the dipeptide pyroglutamyl-alanine. Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensit ivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to t hose of the wild-type Pcp. These residues are not essential for the ca talytic activity. Replacement of Asp-89 by Asn and Ala resulted in enz ymes which retained nearly 25% of activity and which bad no activity, respectively. Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes. Proteins with cha nges of Glu-81 to Gin and Asp-94 to Asn were not detectable in crude e xtract and were probably unstable in bacteria. Our results are consist ent with the proposal that Cys-144 and His-166 constitute the nucleoph ilic and imidazole residues of the Pcp active site, while residue Glu- 81, Asp-89, or Asp-94 might constitute the third part of the active si te, These results lead us to propose Pcps as a new class of thiol amin opeptidases.