Transient kinetic analysis of the catalytic cycle of alkaline phosphatase

Transient kinetic studies were carried out to elucidate the catalytic cycle of Escherichia coli alkaline phosphatase at ph 8.3, 10 degrees C, and to evaluate the rate constants for the individual steps. Using a rapid mixing cell, we were able to detect the burst phase of the reaction, which could not be obtained at alkaline ph values with conventional mixing devices. Analysis of the burst phase revealed that the equilibrium of the initial binding of the substrate, 4-methylumbelliferyl phosphate, to the enzyme is attained rapidly, and that the production of the alcohol, 4-methylumbelliferone, is fast. Analysis of the steady state phase of the reaction yielded a phosphate release rate constant which agrees very well with the kcat determined by initial rate studies. Dephosphorylation of the phosphoryl enzyme prepared at pH 5.7 was studied by the pH-jump technique, using a three-syringe stopped flow apparatus. The results showed that the dephosphorylation step is not rate-limiting in the catalytic cycle and that the presence of substrates or inhibitor has no effect on this step. The lack of effect of substrates on the rate of dephosphorylation and on the rate of phosphate dissociation indicates that the flip-flop mechanism, in which the product release is supposedly facilitated by the binding of a 2nd molecule of substrate, is not valid for alkaline phosphatase. Kinetic constants for various steps in the catalytic cycle of alkaline phosphatase at pH 8.3, 10 degrees C, are reported and a reaction scheme which is in harmony with previous observations from other laboratories is described.