Phosphorylation reaction of vertebrate smooth muscle myosin: an enzyme kinetic analysis

Abstract

Phosphorylation of vertebrate smooth muscle myosin or its isolated 20 000-dalton light chains by myosin light-chain kinase (MLCK) was found to follow first-order kinetics not only at low ([M] much less than Km) but also at high ([M] greater than or equal to Km) substrate concentration. This observation can most simply be explained by a product inhibition for which the Michaelis constants (Km) of the enzyme for the substrate (dephosphorylated myosin) and for the product (phosphorylated myosin) are approximately the same. For such a case, integration of the kinetic velocity equation gives an exponential formula similar to that of a true first-order reaction, the only difference being that its rate constant (k) depends additionally on the initial substrate concentration ([M]0). The standard kinetic constants (k, Km, Vmax) have been calculated by using this pseudo-first-order relationship. Independent evidence for the validity of the derived kinetic relationship was obtained from binding studies with myosin and MLCK. These showed that MLCK binds to phosphorylated and dephosphorylated myosin with approximately equal affinity (Ks = 30 X 10(-9) M). The possible applicability of the same kinetic relationship to other enzyme systems is discussed.