Figure 7. Mode of action toward (a) (d-Phe)2-d-Tyr and (b) d-Tyr-(d-Phe)2: solid circles, d-Tyr; open circles, d-Phe. The reaction mixture (500 iL) containing 2 mM of the substrate (d-Phe)2-d-Tyr or d-Tyr-(d-Phe)2, 50 mM of tris (HCl), pH 9.0, 2 mM of MgSO4, 2% (v/v) of DMSO, and 0.038 unit of the enzyme was incubated at 30 °C. After termination of the reaction, the reaction mixture was analyzed with a Hitachi L-8500 amino acid analyzer.

mase. All three enzymes catalyze the hydrolytic cleavage of the amide bond between D-amino acids of the same configuration. The former two catalyze the transpeptidation reaction; carboxypeptidase DD catalyzes the cross-linkage of the peptideglycan, and D-aminopeptidase catalyzes the transpeptidation and aminolysis of D-amino acid ester and amides, respectively, in water and organic solvents. The aminolysis reaction catalyzed by D-aminopeptidase in bu-tylacetate is efficient (kcat: 7,700/min) (22). The transpep-tidation reaction can also be catalyzed in water, although the product D-alanine-3-aminopentane amide was easily hydrolyzed in water (22). Inheritance of the carboxypep-tidase-like tertiary structure in the D-aminopeptidase would have made the transpeptidation reaction in organic solvents possible. The enzyme is actually inhibited by b-

lactam compounds, such as 6-APA, 7-ACA, benzylpenicil-lin, and ampicillin, although none of these is the substrate for the enzyme.

The D-aminopeptidase is a new member of the penicillin-recognizing enzymes by virtue of the following characteristics: similarities in the primary structure by gene sequencing, similarities in the reactions catalyzed in water and organic solvents, and the findings obtained by kinetic studies of the mutants generated by site-directed mutagenesis and the inhibition by ¡/-lactam compounds. The mutants generated by inhibition of ¡/-lactam compounds may constitute evidence to rebut the contention that the /¡-lactamases evolve from the penicillin-binding proteins (46). The existence of a third enzyme with a similar structure, which does not appear to be a selective target of the / -lactam compounds, was shown. To our knowledge, this enzyme is the first example of an aminopeptidase with Ser at the active site.

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