Substrate‐ and pH‐dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family

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Abstract
Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, kcat/Km, for the modified enzyme decreased by a factor of 8–40 with highly specific substrates, Z‐Gly‐Pro‐Nap, and a fluorogenic octapeptide. With these compounds, the decline in kcat was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc‐Gly‐Pro‐Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (∼500‐fold) in kcat/Km. The second‐order rate constant for the reaction with Z‐Pro‐prolinal, a slow tight‐binding transition‐state analogue inhibitor, and the Kj values for a slow substrate and two product‐like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition‐state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.