Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites.
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 Kis = 6.4 microM 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 d10. Under identical conditions, cadmium is a predominantly competitive inhibitor with Kis = 19.5 microM. Inhibition by mercury is predominantly competitive with Kis = 6.0 microM 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 pKa = 9.2 for cadmium and one pKa 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Wells
T N
TN
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Coulin
F
F
Payton
M A
MA
Proudfoot
A E
AE
D001665:Binding Sites; D001667:Binding, Competitive; D002104:Cadmium / Q000378:metabolism* / Q000494:pharmacology; D002413:Cations, Divalent; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D008433:Mathematics; D008628:Mercury / Q000378:metabolism* / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology*; D008962:Models, Theoretical; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D002413:Cations, Divalent; D008670:Metals; D002104:Cadmium; D008359:Mannose-6-Phosphate Isomerase; D008628:Mercury; D015032:Zinc