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 quantities of the enzyme from the cloned gene. Steady state kinetic studies have been used to characterize the activation by monovalent cations, including Li+, Na+, K+, Rb+, Cs+, Tl+, NH4+, and N(CH3)4+. The enzyme has less than 1% of the maximal activity in the absence of an added monovalent cation, such as K+, Na+, Rb+, Tl+, or NH4+. The enzyme is activated by K+ and Tl+ at lower concentrations than those of other monovalent cations. Li+ and N(CH3)4+ do not activate the enzyme, nor do they inhibit the K(+)-activated enzyme, implying that ionic radius is important in binding selectivity. The Km values for both substrates and Vmax 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, TMP second, and then NAD; the product NADH is released before xanthosine 5'-monophosphate. Substrate and product binding experiments support this mechanism and show the presence of one substrate binding site per subunit. Several rate constants were obtained from a computer simulation of the complete steady state rate equation.
Xiang
B
B
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Taylor
J C
JC
Markham
G D
GD
D001483:Base Sequence; D002414:Cations, Monovalent / Q000494:pharmacology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D007168:IMP Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002414:Cations, Monovalent; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009243:NAD; D007168:IMP Dehydrogenase