Other Cooperativity Models

That the aforementioned cooperativity models are not the only explanations for cooperativity is illustrated by the following brief accounts of other models.

alkaline pH, the rate-limiting step involves a conformational change occurring prior to or during the phosphorylation of the enzyme (Bloch and Schlesinger, 1973). In contrast, utilizing another substrate analogue, O-p-phenylazophenyl phosphorothioate, Chlebowski and Coleman (1972; 1974) reported that alcohol production proceeds with an initial burst and that the amplitude of the burst corresponds to the formation of 2 mol product per dimer at pH 8.5.

When Bale, Chock and Huang (1980b) conducted rapid kinetic experiments on alkaline phosphatase, they were able to demonstrate that catalysis proceeds with an initial burst of alcohol production, followed by a steady-state rate when an organophosphate is used as the substrate. Kinetic analysis of the burst-phase revealed that the equilibrium of the initial binding of the substrate, 4-methyl-umbelliferyl-P, to the enzyme was rapid and that the 4-methyl-umbelliferone formation was fast. (The choice of this substrate is based on the fact that esters of 4-methyl-umbelliferone do not fluo-resce unless the ester linkage is hydrolyzed to form the fluorophore.) Analysis of the steady-state phase of the reaction yielded a Pi-release rate constant that agreed with the kcat determined by initial-rate studies. Dephosphoryl-ation of the phosphoryl enzyme prepared at pH 5.7 was studied by the pH-jump technique, using a three-syringe stopped flow apparatus. The data showed that dephosphory-lation 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

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