KINETIC CHARACTERIZATION OF ENZYME FORMS INVOLVED IN METAL-ION ACTIVATION AND INHIBITION OF MYOINOSITOL MONOPHOSPHATASE

Activation and inhibition of recombinant bovine myo-inositol monophosp hatase by metal ions was studied with two substrates, D,L-inositol 1-p hosphate and 4-nitrophenyl phosphate. Mg2+ and Co2+ are essential acti vators of both reactions. At high concentrations, they inhibit hydroly sis of inositol 1-phosphate, but not 4-nitrophenyl phosphate. Mg2+ is highly selective for inositol 1-phosphate (k(cat.) = 26 s(-1)) compare d with the aromatic substrate (k(cat.) = 1 s(-1)), and follows sigmoid activation kinetics in both cases. Co2+ catalyses the two reactions a t similar rates (k(cat.) = 4 s(-1)), but shows sigmoid activation only with the natural substrate. Li+ and Ca2+ are uncompetitive inhibitors with respect to inositol 1-phosphate, but non-competitive with respec t to 4-nitrophenyl phosphate. Both metal ions are competitive inhibito rs with respect to Mg2+ with 4-nitrophenyl phosphate as the substrate. With inositol 1-phosphate, Ca2+ is competitive and Li+ non-competitiv e with respect to Mg2+. Multiple inhibition studies indicate that Liand P-i can bind simultaneously, whereas no such complex was detected with Ca2+ and P-i. Several sugar phosphates were also characterized as substrates of myo-inositol monophosphatase. D-Ribose 5-phosphate is s lowly hydrolysed (k(cat.) = 3 s(-1)), but inhibition by Li+ is very ef ficient (K-i = 0.15 mM), non-competitive with respect to the substrate and competitive with respect to Mg2+. Depending on the nature of the substrate, Li+ inhibits by preferential binding to free enzyme (E), th e enzyme-substrate (E . S) or the enzyme-phosphate (E . P-i) complex. Ca2+, on the other hand, inhibits by binding to E and E . S, in compet ition with Mg2+. The results are discussed in terms of a catalytic mec hanism involving two metal ions.