Biphasic kinetics induced by modified substrates of penicillinase

The stopped-flow technique was used to examine the initial kinetics of the reaction of penicillinase with benzylpenicillin and with 3 synthetic derivatives of 6-aminopenicillanic acid. While no deviation from linear kinetics was detected with the natural substrate, the slow constant rate of hydrolysis of the derivatives was in each case preceded by an initial, faster rate. The time-dependent transition to the constant rate is reversible and is not due to inhomogeneity of enzyme or substrate preparations. Preincubation with any one derivative eliminates the first phase in the biphasic kinetics of the other 2 derivatives. Since all 3 derivatives are known to distort the active site of penicillinase, the present results demonstrate the capacity of the enzyme to adjust to unfavourable modifications in the substrate.