Chemical and kinetic mechanism of the inositol monophosphatase reaction and its inhibition by Li+
Lithium‐sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21–DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li + ], Li + inhibited the hydrolysis of racemic myo ‐inositol 1‐phosphate, myo ‐inositol 4‐phosphate and adenosine 2′‐phosphate in a linear uncompetitive manner with apparent K i values of 1.1, 0.11 and 1.52 mM, respectively. At Li + concentrations higher than 4 mM, Li + acted as a non‐linear noncompetitive inhibitor for myo ‐inositol 1‐phosphate, K i greater than 1.5 mM. The enzyme was unable to catalyze the transesterification of [ 14 C]inositol in the presence of inositol 1‐phosphate or adenosine 2′‐phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li + , the enzyme was able to release inositol from inositol 1‐phosphate, in a burst, faster than the rate of steady‐state substrate turnover suggesting that Li + binds after P‐O bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of 18 O from [ 18 O] water into phosphate was shown to be completely dependent upon inositol. The K m for inositol for 18 O exchange was 190 mM and in the presence of saturating phosphate, V Ex was at least 60% of V max for the hydrolysis reaction. Thus, the enzyme operates via a ternary‐complex mechanism, and Li + exerts its action by binding to enzyme/product complexes. At low concentration, Li + inhibition with respect to the cofactor, Mg 2+ was non‐competitive. Mg 2+ acted as a non‐competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium‐ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg 2+ with inositol 1‐phosphate and 2′AMP as the substrate but not with inositol 4‐phosphate. The combined results indicate that Mg 2+ and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non‐competitive inhibitor for inositol 1‐phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent ( K i = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li + and phosphate, increasing [Li + ] caused the K i for phosphate to decrease by a factor of (1 + [Li + ]/ K Li ). The K i for the first product off (inositol) was, however, unaltered by Li + . The results indicate that Li + can bind to the species E.Ins.P i and E.P i , but not to enzyme/substrate complexes. Further examination of the burst‐phase release of [ 14 C]inositol and its rate relative to that of the steady‐state reaction under a variety of conditions revealed that Li + acts as a retarder rather than as a dead‐end inhibitor and that the burst was due to hysteresis. Evidence is provided to suggest that Mg 2+ is required for the catalysis only and that Li + occupies the site vacated by Mg 2+ in its action as an inhibitor. The mechanisms of the reactions, the modes of inhibition by Li + and the implications of the finding that inhibition by Li + is enhanced in the presence of phosphate are discussed.