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 [¹⁴C]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 ¹⁸O from [¹⁸O] water into phosphate was shown to be completely dependent upon inositol. The K_m for inositol for ¹⁸O 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, Mg²⁺ was non‐competitive. Mg²⁺ 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 Mg²⁺ with inositol 1‐phosphate and 2′AMP as the substrate but not with inositol 4‐phosphate. The combined results indicate that Mg²⁺ 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 [¹⁴C]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 Mg²⁺ is required for the catalysis only and that Li⁺ occupies the site vacated by Mg²⁺ 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.