Effects of pH and inhibitors on the absorption spectrum of cobalt(II)‐substituted carbonic anhydrase III from bovine skeletal muscle

Abstract

Bovine apocarbonic anhydrase III has been prepared by incubation with 2-c~boxy-l,lO-phenanthroIine at pH 5.5, The Co(II)-substituted enzyme has been prepared and its absorption spectrum has been studied. The spectrum is nearly pH-independent above pH 6. It is very similar to the high pH spectral forms of Co(II)-carbonic anhydrases I and II. The spectra of complexes with the sulfonamide inhibitor, acetazolamide, and with CN-and NCO-are virtually identical to the spectra of the corresponding complexes with Co(II)-isoenzymes I and II. The spectrum of the NS complex indicates that this anion is bound somewhat differently in Co(H) isoenzyme III than in the other Co(II)-substituted isoenzymes. Carbonic anhydrase Isoenzyme Muscle

Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase

“…In fact, the metal ion ligands as well as a number of amino acid residues forming hydrogen bond networks in the active site are conserved in all three isoenzymes [l]. The absorption spectra of the Co(I1)-substituted derivatives are quite similar suggesting that the metal ion coordination geometries are almost identical in isoenzymes I, 11, and 111 [6]. Since the interconversion between C 0 2 and HCO; is thought to take place in the immediate vicinity of the metal ion, it seems reasonable to assume that the essential features of the catalytic mechanism are common to all three isoenzymes, and that the kinetic differences reflect a fine-tuning of the catalytic activity that depends on isoenzyme-specific differences in the active-site structures [ 1, 171.…”

“…The most characteristic, specific feature of the active site of isoenzyme I11 is the presence of some basic amino acid residues, such as Lys-64 and Arg-67, in a hydrophilic part of the active site [l, 181. The positive charges from these residues are thought to stabilize the metal-bound hydroxide ion [5,6,191.…”

“…Carbonic anhydrase 111 was prepared from bovine muscle tissue by ion-exchange chromatography and gel filtration according to method 1 of Engberg et al [2]. Co(I1)-substituted carbonic anhydrase 111 was prepared by adding the chelator 2carboxy-1 ,lo-phenanthroline (synthesized according to Corey et al [7]) to a solution of the enzyme followed by dialysis against an excess of CoClz as described by Engberg and Lindskog [6]. In the following this will be designated 'native' Co(I1)-enzyme.…”

Characterization of carbonic anhydrase from Neisseria gonorrhoeae

“…Co(II)‐substituted NGCA was prepared by adding the chelator 2‐carboxy‐1,10‐phenantroline (synthesized according to Corey et al . [16]) to a solution of the enzyme followed by dialysis against an excess of CoSO 4 as described by Engberg and Lindskog [17]. The excess of CoSO 4 was removed by dialysis against several changes of 50 m m Hepes/NaOH, pH 8.0.…”

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…(7)) is the only or at least a dominant reaction that takes place when sulfonamides bind to a CA, meaning that the intrinsic binding reaction is between the negatively charged sulfonamide to such form of CA where the fourth ligand bound to the Zn II in the active site is electrostatically neutral water molecule. The first indication that the fourth reaction occurs was the spectral evidence on Co-containing CA enzyme (Engberg and Lindskog, 1984; Tu and Silverman, 1986). However, a more recent neutron-diffraction crystal structure of acetazolamide bound to CA II from McKenna laboratory (Fisher et al ., 2012 b ) directly showed that the sulfonamide amino group possesses only one proton and thus the group is deprotonated and negatively charged when bound to the CA II.…”