The pH-dependence and group modification of β-lactamase I

[摘要]

The pH-dependence of the kinetic parameters for the hydrolysis of the β-lactam ring by β-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-α-aminophenylacetamido]penicillanic acid) were used. Both k_cat. and k_cat./K_m for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of k_cat./K_m for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both k_cat. and k_cat./K_m depended on pH in exactly the same way. The value of K_m for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme9s catalytically important groups does not affect binding of this substrate. The pH-dependence of k_cat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward9s reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of β-lactamase I.