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

by Park, Min Sun

Abstract (Summary)
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.
Bibliographical Information:

Advisor:Raushel, Frank M; Lindahl, Paul A; Watanabe, Coran M

School:Texas A&M University

School Location:USA - Texas

Source Type:Master's Thesis



Date of Publication:08/01/2005

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