Transient kinetic studies of pH-Dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance

pH-Dependent kinetic parameters ([k.sub.on], [k.sub.off], and [k.sub.cat]) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme--substrate (ES) complex ([k.sub.on]), the decay of the ES complex ([k.sub.off]), and the formation of the product ([k.sub.cat]) could be analyzed by transient kinetics as mass changes on the QCM plate. The [K.sub.d] ([k.sub.off]/[k.sub.on]) value was different from the Michaelis constant [K.sub.m] calculated from ([k.sub.off] + [k.sub.cat])/[k.sub.on], due to [k.sub.cat] > [k.sub.off]. The rate-determining step was the binding step ([k.sub.on]), and the catalytic rate ([k.sub.cat]) was faster than other [k.sub.on] and [k.sub.off] values. In the range of pH 2.5-5.0, values of [k.sub.on] gradually increased with decreasing pH showing a maximum at pH 3.7, values of [k.sub.off] were independent of pH, and [k.sub.cat] increased gradually with decreasing pH. As a result, the apparent rate constant ([k.sub.cat]/[K.sub.m]) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of [k.sub.on], is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of [k.sub.cat] at the lower pH correlated with the decrease of [alpha]-helix contents of the myoglobin substrate on the QCM.