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To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

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...

It is shown that an "inverse" relationship between the pH dependencies of the rates of hydration of CO2 and dehydration of HCO3-by carbonic anhydrase (EC 4.2.1.1) is a direct consequence of the thermodynamic equilibrium between CO2 and HCO3-and independent of any assumptions about the catalytic mechanism. It is further shown that proposed mechanisms for carbonic anhydrase involving HCO3-as the substrate in the dehydration reaction and a proton transfer reaction, EH+ E + H+, as an obligatory step during catalysis obey the rule of microscopic reversibility. This includes mechanisms in which the proton dissociation is from a zinc-coordinated water molecule. Such mechanisms can be in accord with the observed rapid turnover rates of the enzyme, since rapid proton exchange can occur with the buffer components, EH+ + B = E + BH+. Mechanisms in which H2CO3 is the substrate in dehydration avoid the proton-transfer step, but require that H2CO3 combines with enzyme more rapidly than in a diffusion-controlled reaction. Physicochemical evidence for and against a zinc-hydroxide mechanism is discussed.The zinc metalloenzyme carbonic anhydrase (EC 4.2.1.1) has been investigated by most techniques available for investigation of structure-function relationships (1-4). The crystal structure of the human C isoenzyme has been completed to a resolution of 2 A (2, 3). Yet there is little direct evidence identifying the catalytic and substrate binding groups in the enzyme. It is known that an ionizing group on the enzyme with a pKa near 7 is involved in catalysis, and that the titration of this group results in changes in the immediate environment of the metal ion. As to the identity of this group, the two major proposals are that its basic form represents (a) a zinc-coordinated hydroxide ion and (b) a basic amino-acid side chain, e.g., imidazole, directly or indirectly linked to the metal ion (1, 2, 4). Recently, Koenig and Brown (5) stated that proposed mechanisms involving a zinc-hydroxide as the active group violate the rule of microscopic reversibility, and they postulated that H2CO3 must be the appropriate substrate.As shown in the present paper, the zinc-hydroxide mechanism does not violate microscopic reversibility and, in fact, cannot be ruled out on kinetic or physicochemical grounds. Present data from physicochemical techniques in solution, chemical modification of the enzyme, and x-ray diffraction are analyzed as they pertain to the various proposals for the mechanism of action of carbonic anhydrase. where the equilibrium constants have the following values at 250 (6): Kh = 0.0026; K1 = 10-6 35 M; KHCO = 10-3"77 M.The reversible hydration of CO2 is a relatively slow reaction in the absence of a catalyst; the rate constant for hydration equals about 3.5 X 10-2 sec1.The enzyme-catalyzed reactionThe most active carbonic anhydrases, the human C and the bovine enzymes, catalyze both hydration of C02 and dehydration in the neutral pH range with rate constants between 105 and 106 sec'. This rapid turnover has posed some pr...