MONOVALENT CATION ACTIVATION AND KINETIC MECHANISM OF INOSINE 5'-MONOPHOSPHATE DEHYDROGENASE

Human type II inosine 5' monophosphate dehydrogenase has been purified to homogeneity from an Escherichia coli strain that express large qua ntities of the enzyme from the cloned gene, Steady state kinetic studi es have been used to characterize the activation by monovalent cations , including Li+, Na+, K+, Rb+, Cs+, NH4+ and N(CH3)(4)(+). The enzyme has less than 1% of the maximal activity in the absence of an added mo novalent cation, such as K+, Na+, Rb+, Tl+, or NH4+. The enzyme is act ivated by K+ and Tl+ at lower concentrations than those of other monov alent cations, Li+ and N(CH3)(4)(+) do not activate the enzyme, nor do they inhibit the K+-activated enzyme, implying that ionic radius is i mportant in binding selectivity, The K, values for both substrates and V-max differ with different monovalent cations, Initial velocity and product inhibition kinetic data are consistent with an ordered steady state mechanism in which the enzyme binds K+ first, IMP second, and th en NAD; the product NADH is released before xanthosine 5'-monophosphat e, Substrate and product binding experiments support this mechanism an d show the presence of one substrate binding site per subunit. Several rate constants were obtained from a computer simulation of the comple te steady state rate equation.