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 Ki 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.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (kcat/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo − Fc)φcalc] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.