The Catalytic Mechanism of Carbonic Anhydrase. Hydrogen-Isotope Effects on the Kinetic Parameters of the Human C Isoenzyme

Abstract

1. The steady-state kinetics of the interconversion of CO, and HCO; catalyzed by human carbonic anhydrase C was studied using 'H,O and ' H 2 0 as solvents. The pH-independent parts of the parameters k,,, and K , are 3 -4 times larger in 'H,O than in ,H,O for both directions of the reaction, while the ratios k,,,/K, show much smaller isotope effects. With either C 0 2 or HCO; as substrate the major pH dependence is observed in k,,,, while K , appears independent of pH. The pK, value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2 H 2 0 than in ' H 2 0 .2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhydrase C is approximately 35 faster in 'H20 than in ' H 2 0 . In both solvents the pK, values of the pH-rate profiles are similar to those observed for the C0,-HCO; interconversion.3. It is tentatively proposed that the rate-limiting step at saturating concentrations of C 0 2 or HCO; is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO, and HCO; involves a proton transfer or not.Carbonic anhydrase is a highly efficient catalyst of the reversible interconversion of C 0 2 and HCO,. In a buffered solution not far from neutrality, where Cog-as well as free H + and OH-can be neglected, the stoichiometry of the reaction is CO, + H,O + B e HCO; + BH+, where B and BH' are the basic and acidic buffer components, respectively. Regardless of the specific reaction mechanism, the hydration of C 0 2 must be coupled to the splitting of water, formally into H + and OH-. At some stage of the reaction the OH-ion becomes integrated with C02, while the H + ion ultimately combines with the buffer base. In the reverse reaction OH ~ derived from HCO; must combine with H', originating from the buffer acid, to form H,O. Thus, proton transfers are compulsory ingredients in any mechanism of this reaction.Because of the extremely rapid turnover observed for the enzyme-catalyzed reaction, lo5-lo6 s-' at 25 that H,CO, should be regarded as the substrate species specifically combining with the active site. In effect this means that H + is transported bound to HCO;. It follows from this model that additional proton transfers would have to occur within the enzyme-substrate complex, for example in a concerted reaction as proposed by Kaiser and Lo [3].Arguments against H2C03 as the substrate species combining with the active site have been given by several authors [4-61 pointing out that this would require a second-order rate constant for the binding step exceeding those of diffusion-controlled reactions measured in simpler systems. Alternatively it was suggested that H + is transported between solvent and active site by buffer components acting as proton donors and acceptors. In this case no second-order rate constant involved in the catalytic cycle would have to be greater than lo8-lo9 M-' s -l , and it is not necessary to invoke novel phenomena such as surface diffusion [2,7] to rationalize the e...

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

“…Similar isotope effects on k cat were reported for the bovine CA III ␣-class carbonic anhydrase (45), the ␥-class enzyme Cam (1), and the P. sativum ␤-class carbonic anhydrase (30). The observed solvent hydrogen isotope effect on k cat of 2.1 for Cab is smaller than the value of 3.8 reported for human ␣-class CA II (55), but similar to the value reported for human ␣-class CA IV (27), which follows a mechanism similar to that of CA II.…”

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

“…A conformational change is observed for His-64, the catalytic proton shuttle (Steiner et al, 1975;Tu et al, 1989); the imidazole side chain undergoes a rotation of 58°a bout side chain torsion angle 1 to the "out" conformation (data not shown), as observed in certain CAII structures (e.g. see Nair and Christianson (1991)).…”

The catalytic mechanism of human carbonic anhydrase C: Inhibition of CO2 hydration and ester hydrolysis by HCO−3

“…of 29 300 [6]. 2(N-morpholino)ethanesulfonic acid (MES) and N-2-hydroxethylpiperazine-~-ethanesulfonic acid (HEPES) were obtained from Sigma Chemical Co. Other chemicals were the same as used in previous investigations [2,7]. Stock solutions of CO2 and NaHCOa were prepared as described previously [2].…”

“…The transport of I-1 + between the active site and the solvent is a crucial step in the carbonic anhydrasecatalyzed interconversion of CO2 and HCO~ [1,2]. Recently we reported that the steady-state parameters for both directions of the reaction are subject to substantial hydrogen isotope effects, and we put forward the hypothesis that the rate of catalysis is limited by the transfer of H+between a 'catalytic group' and a 'proton transfer group' [2].…”

“…Recently we reported that the steady-state parameters for both directions of the reaction are subject to substantial hydrogen isotope effects, and we put forward the hypothesis that the rate of catalysis is limited by the transfer of H+between a 'catalytic group' and a 'proton transfer group' [2]. The latter group was assumed to exchange H + rapidly with a buffered medium.…”