In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.

The maximal velocity of catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) requires proton transfer from zinc-bound water to solution assisted by His 64. The catalytic activity of a site-specific mutant of HCA II in which His 64 is replaced with Ala (H64A HCA II) can be rescued by exogenous proton donors/acceptors, usually derivatives of imidazole and pyridine. X-ray crystallography has identified Trp 5 as a binding site of the rescue agent 4-methylimidazole (4-MI) on H64A HCA II. This binding site overlaps with the "out" position in which His 64 in wild-type HCA II points away from the zinc. Activation by 4-MI as proton donor/acceptor in catalysis was determined in the dehydration direction using (18)O exchange between CO(2) and water and in the hydration direction by stopped-flow spectrophotometry. Replacement of Trp 5 by Ala, Leu, or Phe in H64A HCA II had no significant effect on enhancement by 4-MI of maximal rate constants for proton transfer in catalysis to levels near 10(5) s(-1). This high activity for chemical rescue indicates that the binding site of 4-MI at Trp 5 in H64A HCA II appears to be a nonproductive binding site, although it is possible that a similarly effective pathway for proton transfer exists in the mutants lacking Trp 5. Moreover, the data suggest that the out position of His 64 considered alone is not active in proton transfer in HCA II. In contrast to isozyme II, the replacement of Trp 5 by Ala in HCA III abolished chemical rescue of k(cat) by imidazole but left k(cat)/K(m) for hydration unchanged. This demonstrates that Trp 5 contributes to the predominant productive binding site for imidazole, with a maximal level for the rate constant of proton transfer near 10(4) s(-1). This difference in the susceptibility of CA II and III to chemical rescue may be related to the more sterically constrained and electrostatically positive nature of the active site cavity of CA III compared with CA II. The possibility of nonproductive binding sites for exogenous proton donors offers an explanation for the unusually low value of the intrinsic kinetic barrier obtained by application of Marcus theory to chemical rescue of H64A HCA II.

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We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His 7 in HCA III. Using an 18 O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His 7 and His 64 within the active site cavity, shows the conformation of the side chain of His 64 moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His 7 . This suggests a cause of decreased proton transfer in catalysis.The carbonic anhydrases in the ␣ class include the mammalian isozymes and are all zinc-containing monomeric enzymes, generally with molecular masses near 30 kDa. These isozymes of carbonic anhydrase catalyze the dehydration/hydration of HCO 3 Ϫ /CO 2 by a two-stage or ping-pong mechanism in which the first step (Equation 1) is the reaction of bicarbonate with the enzyme containing zinc-bound water resulting in the formation of CO 2 and leaving zinc-bound hydroxide at the active site (1, 2).The second stage is the regeneration of the zinc-bound water through proton transfer (1, 2). BH ϩ represents a proton donor that is a residue of the enzyme itself or buffer in solution. In the carbonic anhydrases, the maximal velocities of catalysis and the rates of exchange of 18 O between CO 2 and water are limited by the intramolecular proton transfers indicated in Equation 2. This was initially determined by observation of a solvent deuterium isotope effect of 3.8 on k cat for hydration catalyzed by human carbonic anhydrase (HCA) 1 II, among the most efficient isozymes in the ␣ class of carbonic anhydrases (3). The solvent hydrogen isotope effect was 2.4 for 18 O exchange catalyzed by HCA III, the least efficient of the ␣ class (4). Subsequent results including pH profiles, buffer activation, and computer simulations for these isozymes support the rate-limiting nature of proton transfer in these isozymes (4 -7).For HCA II, the catalytic turnover is near 10 6 s Ϫ1 . For this isozyme the predominant shuttle residue has been identified as His 64 (3, 5), the side chain of which extends into the active site cavity without apparent interactions with other residues of the protein (8, 9). The imidazole ring of His 64 is about 7.5 Å from the zinc when this side chain is in the "in" conformation pointing toward the metal (8). This is too far for direct proton transfe...