Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana beta-carbonic anhydrase provide direct evidence for the role of this residue in beta-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal k(cat) (33% of wild type) at steady state and the maximal rate of CO(2)(-) HCO(2)(-) exchange at chemical equilibrium as measured by R(1)/[E] (7% of wild type), while leaving the maximal rate of H(+) transfer, as measured by k(cat) at steady state, or R(H(2)O)) at chemical equilibrium, largely unaffected.     

Proton transfer within the active-site cavity of carbonic anhydrase III

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.     

Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II

Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.     

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Chemical rescue in catalysis by human carbonic anhydrases II and III

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.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

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.     

Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II

Catalysis of the hydration of CO2 by human carbonic anhydrase isozyme II (HCA II) is sustained at a maximal catalytic turnover of 1 mus-1 by proton transfer between a zinc-bound solvent and bulk solution. This mechanism of proton transfer is facilitated via the side chain of His64, which is located 7.5 A from the zinc, and mediated via intervening water molecules in the active-site cavity. Three hydrophilic residues that have previously been shown to contribute to the stabilization of these intervening waters were replaced with hydrophobic residues (Y7F, N62L, and N67L) to determine their effects on proton transfer. The structures of all three mutants were determined by X-ray crystallography, with crystals equilibrated from pH 6.0 to 10.0. A range of changes were observed in the ordered solvent and the conformation of the side chain of His64. Correlating these structural variants with kinetic studies suggests that the very efficient proton transfer (approximately 7 micros-1) observed for Y7F HCA II in the dehydration direction, compared with the wild type and other mutants of this study, is due to a combination of three features. First, in this mutant, the side chain of His64 showed an appreciable inward orientation pointing toward the active-site zinc. Second, in the structure of Y7F HCA II, there is an unbranched chain of hydrogen-bonded waters linking the proton donor His64 and acceptor zinc-bound hydroxide. Finally, the difference in pKa of the donor and acceptor appears favorable for proton transfer. The data suggest roles for residues 7, 62, and 67 in fine-tuning the properties of His64 for optimal proton transfer in catalysis.     

Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal beta-class carbonic anhydrase

The roles of an aspartate and an arginine, which are completely conserved in the active sites of beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the beta-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k(cat)/K(m) value depends only on the rate constants for the CO(2) hydration step, whereas k(cat) also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k(cat)/K(m) values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO(2) hydration step. Parallel decreases in k(cat) and k(cat)/K(m) values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k(cat) of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k(cat) and k(cat)/K(m) values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k(cat) of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.     

Crystal structure of F65A/Y131C-methylimidazole carbonic anhydrase V reveals architectural features of an engineered proton shuttle

The crystal structure of F65A/Y131C murine alpha-carbonic anhydrase V (CAV), covalently modified at cysteine residues with 4-chloromethylimidazole, is reported at 1.88 A resolution. This modification introduces a methylimidazole (MI) group at residue C131 in the active site with important consequences. F65A/Y131C-MI CAV exhibits an up to 3-fold enhancement of catalytic activity over that of wild-type CAV [Earnhardt, J. N., Wright, S. K., Qian, M., Tu, C., Laipis, P. J., Viola, R. E., and Silverman, D. N. (1999) Arch. Biochem. Biophys. 361, 264-270]. In this modified CAV variant, C131-MI acts as a proton shuttle, facilitating the deprotonation of a zinc-bound water molecule to regenerate the nucleophilic zinc-bound hydroxide ion. A network of three hydrogen-bonded water molecules, across which proton transfer likely proceeds, bridges the zinc-bound water molecule and the C131-MI imidazole group. The structure of F65A/Y131C-MI CAV is compared to structures of Y64H/F65A murine CAV, wild-type human alpha-carbonic anhydrase II, and the gamma-carbonic anhydrase from Methanosarcina thermophilain an effort to outline common features of catalytic proton shuttles.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.     

Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila.

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.     

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.     

The catalytic mechanism of carbonic anhydrase. Hydrogen-isotope effects on the kinetic parameters of the human C isoenzyme.

1. The steady-state kinetics of the interconversion of CO2 and HCO3 catalyzed by human carbonic anhydrase C was studied using 1H2O and 2H2O as solvents. The pH-independent parts of the parameters k(cat) and Km are 3-4 times larger in 1H2O than in 2H2O for both directions of the reaction, while the ratios k(cat)/Km show much smaller isotope effects. With either CO2 or HCO3 as substrate the major pH dependence is observed in k(cat), while Km appears independent of pH. The pKa value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2H2O than in 1H2O. 2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhudrase C is approximately 35% faster in 2H2O than in 1H2O. In both solvents the pKa values of the pH-rate profiles are similar to those observed for the CO2-HCO3 interconversion. 3. It is tentatively proposed that the rate-limiting step at saturating concentrations of CO2 or HCO3 is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO2 and HCO3 involves a proton trnasfer or not.     

Characterization of carbonic anhydrase from Neisseria gonorrhoeae.

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO(2) hydration and the similar behaviour of the kinetics of (18)O exchange between CO(2) and water at chemical equilibrium. The pH profile of the turnover number, k(cat), can be described as a titration curve with an exceptionally high maximal value of 1.7 x 10(6) s(-1) at alkaline pH and a pK(a) of 7.2. At pH 9, k(cat) is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio k(cat)/K(m) is dependent on two ionizations with pK(a) values of 6.4 and 8.2. However, an (18)O-exchange assay identified only one ionizable group in the pH profile of k(cat)/K(m) with an apparent pK(a) of 6.5. The results of a kinetic analysis of a His66-->Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar K(i) values for the inhibitors NCO(-), SCN(-) and N(3)(-) as HCA II, while CN(-) and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO(-), SCN(-), CN(-) and N(3)(-) resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO(2) hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A(292)/A(260) ratio was not affected by the presence of TCEP, and a structural transition at 2.8--2.9 M GdnHCl was observed.     

Roles of substrate distortion and intramolecular hydrogen bonding in enzymatic catalysis by scytalone dehydratase.

Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstraction from its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by approximately 2 kcal/mol. A survey of 12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for DDBO over scytalone and for DDBO over vermelone than the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8 hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond of vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for vermelone over deshydroxyvermelone for the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition and catalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermelone and vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzyme catalysis.     

Cloning, expression and some properties of alpha-carbonic anhydrase from Helicobacter pylori

The alpha-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25 degrees C are kcat=2.4x10(5) s(-1), KM=17 mM and kcat/KM=1.4x10(7) M(-1) x s(-1). The pH dependence of kcat/KM fits with a simple titration curve with pK(a)=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k(enz), of 24 M(-1) x s(-1) at pH 8.8 and 25 degrees C. However, with 2-nitrophenyl acetate as substrate a k(enz) value of 665 M(-1) x s(-1) was obtained under similar conditions.     

Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase

The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.     

A 13C nuclear magnetic resonance study of CO2/HCO-3 exchange catalyzed by human carbonic anhydrase I

Rates of CO2/HCO-3 exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of 13C-labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7-8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 1H2O or 2H2O as solvents. Chloride ions inhibit CO2/HCO-3 exchange competitively. The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme II (or C). From a comparison of the exchange kinetics with the steady-state kinetics of CO2 hydration and HCO-3 dehydration it is tentatively concluded that the transfer of H+ between active site and medium proceeds with rates of similar magnitudes in the two isoenzymes, whereas the central catalytic step, the interconversion of enzyme-bound CO2 and HCO-3, is much slower in isoenzyme I than in isoenzyme II.