Comparative studies of bovine and human B carbonic anhydrases through their cobalt(II) substituted derivatives

Abstract

A major physicochemical difference between the high activity bovine carbonic anhydrase B (BCAB) and the low activity human B isoenzyme (HCAB) resides in the pK a s of the groups controlling the catalytic activity, which differ by at least one unit. Such a difference is maintained in the cobalt(II) substituted derivatives. The electronic spectra of the latter show in both cases a pH dependence which can be rationalized in terms of at least two acidic groups with close pK a values [1]. Studies on the bovine isoenzyme and its adducts with inhibitors which act as metal ligands allowed us to propose a spectroscopic criterion to assign the coordination number of the metal, based on the combined use of electron spectroscopy, water proton NMRD, and 1H NMR of the coordinated histidines [2–4]. With such a background, we turned to the investigation of the human B isoenzyme with the aim of giving the observed differences a more firm structural basis. A careful examination of the electronic spectra of the low pH form of the latter derivative shows that the molar absorbance is considerably lower than that of the corresponding bovine isoenzyme; NMRD measurements extended down to 0.01 MHz indicate a substantially lower water proton relaxation capability of the chromophore [5], while T 1 measurements of the 4H signal of the coordinated His 119 show that the lower nuclear relaxing efficiency is due to a shorter electronic relaxation time [6]. All of these data are taken as evidence for a large percentage of five coordinate cobalt(II) in the low pH form of the human isoenzyme. The metal donor set would then be constituted by three histidine nitrogens and two water oxygens. There are convincing experimental results and arguments to indicate that the main activity-linked acid base group is a coordinated water molecule. The existence of five coordinated chromophore in the low pH form of CoHCAB, as opposed to the mainly four-coordinate CoBCAB, for the first time satisfactorily accounts for the difference in pK a of the coordinated water. CoBCAB and CoHCAB are further differentiated by the higher affinity for imidazole and related ligands of the human isoenzyme [7]. The pH dependence of the affinity of such ligands for the enzyme is accounted for. At low pH the behavior of the two isoenzymes towards anionic inhibitors appears to be different. While the spectra of the adducts with CoBCAB are pH insensitive in the range of existence of the complexes, the human isoenzyme shows an increase in molar absorbance at low pH. Such behavior is shown particularly by thiocyanate. Whereas the affinity of the inhibitor for the isoenzyme is governed by a pK a of ∼7.5, that of the change in molar absorbance is governed by a pK a of 6.5. Such differences are reminiscent of the different behavior of the copper derivatives with respect to sulfonamides [8].