Kinetic characterization of enzyme forms involved in metal ion activation and inhibition of myo-inositol monophosphatase.
Activation and inhibition of recombinant bovine myo-inositol monophosphatase by metal ions was studied with two substrates, D,L-inositol 1-phosphate and 4-nitrophenyl phosphate. Mg2+ and Co2+ are essential activators of both reactions. At high concentrations, they inhibit hydrolysis of inositol 1-phosphate, but not 4-nitrophenyl phosphate. Mg2+ is highly selective for inositol 1-phosphate (kcat. = 26 s-1) compared with the aromatic substrate (kcat. = 1 s-1), and follows sigmoid activation kinetics in both cases. Co2+ catalyses the two reactions at similar rates (kcat. = 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 respect to 4-nitrophenyl phosphate. Both metal ions are competitive inhibitors with respect to Mg2+ with 4-nitrophenyl phosphate as the substrate. With inositol 1-phosphate, Ca2+ is competitive and Li+ non-competitive with respect to Mg2+. Multiple inhibition studies indicate that Li+ and Pi can bind simultaneously, whereas no such complex was detected with Ca2+ and Pi. Several sugar phosphates were also characterized as substrates of myo-inositol monophosphatase. D-Ribose 5-phosphate is slowly hydrolysed (kcat. = 3 s-1), but inhibition by Li+ is very efficient (Ki = 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), the enzyme-substrate (E.S) or the enzyme-phosphate (E.Pi) complex. Ca2+, on the other hand, inhibits by binding to E and E.S, in competition with Mg2+. The results are discussed in terms of a catalytic mechanism involving two metal ions.
Strasser
F
F
Marion Merrell Dow Research Institute, Strasbourg, France.
Pelton
P D
PD
Ganzhorn
A J
AJ
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D004789:Enzyme Activation; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D008670:Metals; D009596:Nitrophenols; C002887:bis(4-nitrophenyl)phosphate; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase