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Biochemistry. 1993 Mar 30;32(12):3058-66.

Thermodynamics of binding of the CO2-competitive inhibitor imidazole and related compounds to human carbonic anhydrase I: an isothermal titration calorimetry approach to studying weak binding by displacement with strong inhibitors.

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1
Department of Biochemistry and Molecular Biology, University of Kansas School of Medicine, Missouri.

Abstract

The visible spectrum of Co(II)-substituted human carbonic anhydrase I (HCA I) complexed with the unique CO2-competitive inhibitor imidazole undergoes a marked alkaline intensification, with a midpoint near pH 8 [Bauer, R., Limkilde, P., & Johansen, J. T. (1977) Carlsberg Res. Commun. 42, 325-339]. This change was first attributed to the ionization of a nondisplaced water ligand of the active-site metal in a five-coordinate complex. Later proposals favored assigning it to the deprotonation of the bound imidazole itself to give a tetrahedrally coordinated imidazolate anion at high pH. We have determined by isothermal titration calorimetry the pH dependence of the enthalpy of binding of imidazole and its analogues to HCA I and Co(II)HCA I. We devised an indirect strategy whereby the enthalpy of binding of the strong sulfonamide inhibitor methazolamide was determined in the absence and presence of a constant high concentration of the competing imidazole or its analogues. The standard enthalpy of binding of deprotonated methazolamide to the "acid" form of HCA I and Co(II)HCA I was found to be pH independent over the pH range of 6.5-9.5, as expected. It was also identical for both the zinc (-13.5 +/- 1.1 kcal M-1) and the cobalt (-13.7 +/- 0.4 kcal M-1) forms. The standard enthalpy of binding of neutral imidazole (average value -6.1 +/- 0.8 kcal M-1) surprisingly did not show any marked pH dependence, varying by about 1.1 and 2.6 kcal M-1 for the zinc and cobalt enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS).

PMID:
8457566
[Indexed for MEDLINE]
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