Kinetic behaviour of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate

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Abstract
The effects of varying pH, ionic strength and temperature on the parameters Km and Vmax for a purified alkaline phosphatase from calf intestinal mucosa with a new fluorogenic substrate, 4-methylumbelliferyl phosphate monoester disodium salt, and an ammediol-hydrochloric acid buffer system were determined. It was found that, under varying conditions, a relationship exists between Km and Vmax, such that Vmax =[beta]/(1 + [alpha]/Km), where a and[beta] are constants, temperature- and ionic strength-dependent, but pH-independent. It is shown that this relationship accounts satisfactorily for the well-known effect of varying substrate concentration on optimum pH and velocity. The various results are interpreted in terms of a pH-dependent conformational equilibrium between 2 forms of the enzyme, E1 and E2. Only E1 combines with substrate, and only E2 reacts to give inorganic phosphate. To account for the pH-variation of Km and Vmax in terms of this theory, it is postulated that the conformational change is associated with a change in pK of 2 basic groups in the enzyme.