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

Abstract

Phosphorylation of vertebrate smooth muscle myosin or its isolated 20,00-dalton L chains by myosin L-chain kinase (MLCK) was found to follow 1st-order kinetics not only at low ([M] .mchlt. Km) but also at high ([M] .gtoreq. Km) substrate concentration. This observation can most simply be explained by a product inhibition for which the 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 1st-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) were calculated by using this pseudo-1st-order relationship. Independent evidence for the validity of the derived kinetic relationship was obtained from binding studies with myosin and MLCK. MLCK binds to phosphorylated and dephosphorylated myosin with approximately equal affinity (Ks = 30 .times. 10-9 M). The possible applicability of the same kinetic relationship to other enzyme systems is discussed.