Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites

• Published in: Biochimica et biophysica acta Vol. 32; no. 5; pp. 1294 - 1301
• Main Authors: Wells, T.N.C, Coulin, F, Payton, M.A, Proudfoot, A.E.I
• Format: Journal Article
• Language: English
• Published: 1993
• Subjects: binding sites; cadmium; enzyme activity; inhibition; isomerases; mannose; mannose 6-phosphate; mercury; Saccharomyces cerevisiae; sugar phosphates; zinc
• ISSN: 0006-3002; 1878-2434

Phosphomannose isomerase (PMI) from Saccharomyces cerevisiae is a zinc-dependent metalloenzyme. Besides its role in catalysis, zinc is also a potent inhibitor of the enzyme. The inhibition is competitive with the substrate mannose 6-phosphate, with K(is) = 6.4 micromolar in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees C. This inhibition constant is 4 orders of magnitude smaller than for group II divalent cations, indicating that the binding is not primarily electrostatic. Micromolar inhibition is also observed with ions of the other metals of the electronic configuration d(10). Under identical conditions, cadmium is a predominantly competitive inhibitor with K(is) = 19.5 micromolar. Inhibition by mercury is predominantly competitive with K(is) = 6.0 micromolar but shows a hyperbolic Dixon plot. Theorell-Yonetani double-inhibition analysis shows that zinc and cadmium ions are mutually exclusive inhibitors against mannose 6-phosphate. However, analysis of zinc and mercury double inhibition shows that they can simultaneously bind in the mannose 6-phosphate binding pocket, with only a small mutual repulsion. Inhibition of the enzyme by cadmium and zinc ions is strongly pH dependent with pK(a) = 9.2 for cadmium and one pK(a) at 6.6 and two at 8.9 for zinc. The inhibitory species are the monohydroxide forms, Zn(OH)+ and Cd(OH)+. However, inhibition by mercury is relatively pH-independent, consistent with the neutral Hg(OH)2 being the inhibitory species. In all three cases, the metal ion binding causes a conformational change in the enzyme as judged by tryptophan fluorescence. The K(s) at pH 8.0 determined by fluorimetry is similar to the kinetic K(i) for zinc and cadmium but is 5-fold higher for mercury. These studies demonstrate that the zinc triad of metal ions specifically inhibits phosphomannose isomerase. However, although all three metals are inhibitors with respect to the mannose 6-phosphate substrate, there are two distinct modes of inhibition, one for zinc and cadmium and the other for mercury.