UNSPECIFIED. (2002) Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase. JOURNAL OF BIOLOGICAL CHEMISTRY, 277 (47). pp. 44597-44605. ISSN 0021-9258

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Abstract

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp 641 residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K-m for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K-m. This indicates that the contribution of Asp641 is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH2Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry
Journal or Publication Title:	JOURNAL OF BIOLOGICAL CHEMISTRY
Publisher:	AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
ISSN:	0021-9258
Date:	22 November 2002
Volume:	277
Number:	47
Number of Pages:	9
Page Range:	pp. 44597-44605
Identification Number:	10.1074/jbc.M207386200
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/10368

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