Catalytic properties and mechanism studies of the PepQ prolidase from Escherichia coli



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Texas A&M University


The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with proline residues 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 x 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 was assessed as substrates for this enzyme. 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 more slowly hydrolyzed 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. The pH-rate profiles for the wild-type E. coli prolidase using proline dipeptides as substrates were obtained. The roles of H346, H228, and E384 in the enzyme catalytic mechanism were also investigated by obtaining the pH-rate profiles for the mutants H346N, H228N, and E384Q. In an effort to clarify the mechanistic role of the interaction of the ????-amino group of Xaa-Pro with metal at the enzyme active site, comparisons of the hydrolytic activity for Ala-Pro and 1-(1-oxopropyl)-L-proline, in which a hydrogen replaces the ????-amino group of Ala-Pro, were performed.