Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase.
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 Asp(641) 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-CH(2)Cl 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.
Szeltner
Zoltán
Z
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518, P. O. Box 7, Budapest 112, Hungary.
Rea
Dean
D
Juhász
Tünde
T
Renner
Veronika
V
Mucsi
Zoltán
Z
Orosz
György
G
Fülöp
Vilmos
V
Polgár
László
L
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D013552:Swine
D007527:Isoenzymes; D010455:Peptides; D001224:Aspartic Acid; D012965:Sodium Chloride; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases