Nuclear Magnetic Resonance Studies of Carbonic Anhydrase. Binding of Sulfacetamide to the Manganese Enzyme

Amos Lanir, Gil Navon

Research output: Contribution to journalArticlepeer-review

Abstract

The effect of manganese(II)-bovine carbonic anhydrase on the proton magnetic relaxation times T1 and T2 of sulfacetamide was studied. The activity and stability of the manganese-enzyme complex were determined. The specificity of sulfacetamide binding to the active site was demonstrated by its replacement by p-toluenesulfonamide and azide ion. The equilibrium constant for the inhibitor-enzyme complex determined by the nuclear magnetic resonance method was found to agree with that obtained by kinetic measurements, providing further evidence for the specificity of the binding. The enhancement of the relaxation rate, 1/T2, appeared to be controlled by both the exchange lifetime and the dipolar relaxation mechanisms. Their relative contributions were separated using the measured T1/T2 ratios and the correlation time for the dipolar interaction could be determined. It was found that the electronic spin relaxation is the dominating correlation time at lower temperatures, while the rotational correlation time, which has a different temperature dependence, dominates at higher temperatures. Substituting the correlation times in the Solomon-Bloembergen equations for relaxation rates of the bound inhibitor, the distances between the manganese ion and protons of the methyl group, and phenyl protons ortho and meta to the sulfonamide group in the bound inhibitor molecule were calculated as 4.6 ± 0.2, 5.6 ± 0.3, and 6.6 ± 0.4 Å, respectively. These distances fit with a model in which the sulfonamide nitrogen is directly bound to the metal ion. However, this model is not unique. From the exchange lifetime and the equilibrium binding constants, assuming one-step inhibition mechanism, the rate constants for the association and dissociation reactions and their temperature dependences were derived.

Original languageEnglish
Pages (from-to)3536-3544
Number of pages9
JournalBiochemistry
Volume11
Issue number19
DOIs
StatePublished - 1 Sep 1972

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