Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The kcat and kcat/Km values for the hydrolysis of Met-Pro are 109 s−1 and 8.4 × 105 M−1 s−1, respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The SP-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a kcat of 36 min−1 and a kcat/Km of 710 M−1 s−1. The corresponding RP-enantiomer was hydrolyzed more slowly with a kcat of 0.4 min−1 and a kcat/Km of 11 M−1 s−1. The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.