The determination of the binding constant of metalloenzymes for their active site metal ion from ligand inhibition data: Theoretical analysis and application to the inhibition of thermolysin by 1,10-phenanthroline |
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Authors: | Gerrit Voordouw Carol Milo Rodney S. Roche |
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Affiliation: | Biopolymer Research Group, Department of Chemistry, The University of Calgary, Calgary T2N 1N4, Alberta, Canada |
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Abstract: | An equation is found relating the fractional activity, (v/v0), of an enzyme assay mixture to the total concentrations of metalloenzyme, active site metal ion, metal-binding ligand and substrate and the stability constants of the complexes present. When (v/v0) is measured as a function of the total ligand concentration, this equation offers a way of data-plotting which yields straight lines and permits the calculation of the metal-binding constant KME from either the slope or the intercept, provided that mixed complexes (enzyme-metal ion-ligand) do not contribute significantly to the change in (v/v0). Since deviations from linearity occur in the latter case, the proposed inhibition plot serves as a diagnostic tool for the recognition of such complexes. Application to the inhibition of thermolysin by 1,10-phenanthroline gives a value of 2.1 × 1011m−1 for KZnE, the binding constant of the active site zinc ion, at pH 7.50, 25°C and ionic strength 0.1. The equation also allows the rapid calculation of the ligand concentration necessary to attain a desired degree of inhibition when the total enzyme and active site metal ion concentrations of the solution are known. |
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Keywords: | Author to whom correspondence should be addressed. |
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