Chemical and kinetic mechanism of the inositol monophosphatase reaction and its inhibition by Li+

Andrew P. Leech, Graham R. Baker, Janis K. Shute, Mark A. Cohen, David Gani*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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 Ki 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, Ki 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 Km for inositol for 18O exchange was 190 mM and in the presence of saturating phosphate, VEx was at least 60% of Vmax 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 (Ki= 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 Ki for phosphate to decrease by a factor of (1 + [Li+]/KLi). The Ki for the first product off (inositol) was, however, unaltered by Li+. The results indicate that Li+ can bind to the species E.Ins.Pi and E.Pi, 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.

Original languageEnglish
Pages (from-to)693-704
Number of pages12
JournalEuropean Journal Of Biochemistry
Volume212
Issue number3
DOIs
Publication statusPublished - Mar 1993

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