Figure 6. Time course of (d-Phe)4 hydrolysis by d-aminopepti-dase. The reaction mixture consisted of 2 mM of (d-Phe)4, 100 mM of tris (HCl), pH 9.0, 2 mM of MgSO4, 2% (v/v) of DMSO, and 0.038 unit of the enzyme solution in a total volume of 500 iL. The reaction was carried out at 30 °C and terminated with HClO4. The amounts of (d-Phe)4 (circles), (d-Phe)2 (squares), and d-Phe (triangles) in the supernatant were measured by a Waters HPLC equipped with a Cosmosil 5C18-MS at a flow rate of 1.0 mL/min with a mixture of methanol (55%) and 5 mmol KH2PO4/H3PO4, pH 2.9 (45%).

lactamase is that the consensus sequence is located around 60 from the N-terminus of the enzymes (43).

The inhibition by p-chloromercuribenzoate (PCMB) and other sulfhydryl reagents suggested that the enzyme would be a thiol peptidase (44). However, the findings of the site-specific mutagenesis study showed that the amino acid sequences Ser-Xaa-Xaa-Lys, which had been conserved in the penicillin-recognizing enzymes, are essential in exerting the D-aminopeptidase activity (28). The sites Ser-61 and Lys-64 are essential in the catalysis, because the Vmax/ Km value measured with the mutants modified to Cys-61 and Asn-64 have been reduced to 0.26% and 0.008%, respectively, of those of the natural enzyme, although these Km values were hardly lowered. The mutant with the inert Gly residue in place of the likely active center Ser-61 had a lower activity (10~5-fold that of the native enzyme). On the other hand, the mutations at other sites (Met-57 and Cys-68) did not greatly affect the enzyme activity. The mutations at Cys-60, which is adjacent to the likely active center Ser-61, gave notable effects on the kinetic profiles, indicating that the residue is important in the binding of the substrate. The substitutions at Cys-60 to Ser and Gly produced mutants with slightly altered Vmax/Km values. They were tolerant to inhibition by PCMB, which suggests that the inhibition of the native enzyme by PCMB would have been due to the steric hindrance by a mercaptide bond formation between Cys-60 of the enzyme and PCMB.

A mutant of D-aminopeptidase with increased thermo-stability was obtained. The enhancement of the thermal stability of mutant enzyme was attributed to the substitution of Gly-155 to Ser (45).

Figure 5 shows similarities of the reactions catalyzed by D-aminopeptidase, carboxypeptidase DD, and /-lacta-

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