Y. Pocker

Active‐Site Mechanisms of the Carbonic Anhydrasesa

During the last twenty years, carbonic anhydrase (CA) (EC 4.2.1.1) has played a significant role in the continuing illumination of the principles underlying enzyme activity. In addition to its intrinsic physiological importance, CA is an extremely convenient enzyme for study. The isolated zinc metalloenzyme is stable in solution and during storage; in fact, the highly active form, CA 11, can tolerate pH values in the range 5.5 to 12. As a result of its pronounced catalytic power and robust constitution, CA was transformed into a veritable “laboratory” in which enzyme catalysis was rigorously tested and explored employing the numerous principles of solution chemistry.

Mechanistic aspects of coordination, catalysis and control in carbonic anhydrase

Abstract Among the most significant developments in the bioinorganic chemistry of carbonic anhydrase during the past few years are those associated with the role of coordinated zinc(II) ion in the activation of H2O and bicarbonate. Illustrative of the catalytic processes encompassed by these developments are many hydration-dehydration processes and a variety of hydrolysis reactions. Contributing to the intensive interest and research that this field has generated is the unusual catalytic efficiency connected with the physiologically important process, eqn. 1: CO2 + H2O ⇄ H+ + HCO−3 The problems of elucidating the details of the enzymatic pathway are compounded not only by its multistep character but also by the fact that each of the intermediates in the proposed catalytic cycle coexists in several forms related not only through proton transfers but also via ligand addition and dissociation processes. Substitution of an alkyl group (methyl through n-pentyl) for the proton of bicarbonate dramatically alters the properties of the resultant alkyl carbonate esters, ROCO−2, toward the enzyme. While bicarbonate is the natural substrate of various carbonic anhydrases with turnover numbers of ∼106 s−1, the alkyl carbonates show no detectable activity as substrates. The alkyl carbonates, however, bind efficiently to the various carbonic anhydrases and act as typical anionic inhibitors of enzyme catalyzed CO2 hydration and HCO−3 dehydration, with Ki values comparable to those of the corresponding RCO−2 anions. It appears that the substitution of an alkyl group inhibits a proton transfer essential in the enzyme-catalyzed dehydration of HCO−3, and further that the bicarbonate proton permits a unique binding interaction with carbonic anhydrase. Examination of the kinetic features (temperature-jump, stopped-flow and NMR) of the catalytic system emphasizes a number of additional points, notably: (a) The requirement of several binding sites. (b) Non-protein ligand lability during the interconversion of ES, EP and ESP. (c) Accessibility of two different coordination numbers for ZN(II)-, Co(II)- and Mn(II)-carbonic anhydrases. (d) Accessibility of two or more protonation states in the active site. (e) The potentially important role of the ‘spectator’ ligands (i.e. the three histidyl residues) in regulating catalytic activity and selectivity. The results we present and the mechanism we propose provide an appealing structural model for carbonic anhydrase catalysis. The implications of these labile structures lead to fruitful mechanistic insights into a number of kinetic observations on this fascinating catalyst and provide some guidance in the search for the catalytic mechanism of carbonic anhydrase.

Metalloenzymes and Model Systems. Carbonic Anhydrase: Solvent and Buffer Participation, Isotope Effects, Activation Parameters and Anionic Inhibition

The interconversion between CO2 and HCO 3 - in the presence of bovine carbonic anhydrase (BCA) was studied by initial rate measurements using a stopped-flow indicator method. The results analyzed in terms of the Michaelis-Menten Scheme exhibited a sigmoidal variation of the turnover number (kcat) against pH with a pKa value of about 6.8 at 25.0°. For CO2 hydration large kcat values were observed in the high pH region, while for HCO 3 - dehydration large kcat values were observed in the low pH region. For both substrates Km values did not show any significant variation with pH in the region studied. Solvent deuterium isotope effects were found to be between 2.5 and 3.0 at 25.0° for both kcat and Km, and did not vary significantly with change of pH or substrate. Activation parameters for CO2 hydration catalyzed by H2O, OH-, and BCA were obtained in the temperature range 7.0°–35.0°C. For BCA catalysis, temperature effects were confined largely to kcat. Inhibition of BCA by monoanions was studied over a pH range of 6.6 to 9.0 for CO2 hydration, and 6.6 to 7.0 for HCO 3 - dehydration. Anions were found to exhibit linear mixed inhibition of CO2 hydration at low pH, linear uncompetitive inhibition of CO2 hydration at high pH, and linear competitive inhibition of HCO 3 - dehydration at all pH values studied. The implications of these results are discussed, a formal kinetic scheme is proposed, and a mechanism presented to account for these observations.