Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase.
On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments. Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the 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 sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp. These residues are not essential for the catalytic activity. Replacement of Asp-89 by Asn and Ala resulted in enzymes which retained nearly 25% of activity and which had no activity, respectively. Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes. Proteins with changes of Glu-81 to Gln and Asp-94 to Asn were not detectable in crude extract and were probably unstable in bacteria. Our results are consistent with the proposal that Cys-144 and His-166 constitute the nucleophilic 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 site. These results lead us to propose Pcps as a new class of thiol aminopeptidases.
Le Saux
O
O
Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires Institut National des Sciences Appliquees, Villeurbanne, France.
Gonzales
T
T
Robert-Baudouy
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D011751:Pyroglutamyl-Peptidase I