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

Abstract

1. The effects of varying pH, ionic strength and temperature on the parameters K(m) and V(max.) 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. 2. It was found that, under varying conditions, a relationship exists between K(m) and V(max.) such that V(max.)=beta/(1+alpha/K(m)), where alpha 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. 3. The various results are interpreted in terms of a pH-dependent conformational equilibrium between two forms of the enzyme, E(1) and E(2). Only E(1) combines with substrate, and only E(2) reacts to give inorganic phosphate. 4. To account for the pH-variation of K(m) and V(max.) in terms of this theory, it is postulated that the conformational change is associated with a change in pK of two basic groups in the enzyme.