A 13C nuclear-magnetic-resonance study of CO2-HCO3-exchange catalyzed by human carbonic anhydrase C at chemical equilibrium.
The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of 13CO2 and NaH13CO3 were measured at 67.89 MHz. Enzyme-catalyzed CO2-HCO-3 exchange rates were estimated from the linewidths of the resonances. The results show that:(a) the maximal exchange rates are larger than the maximal turnover rates;(b) the exchange is equally rapid with 1H2O or with 2H2O as solvents;(c) the exchange is equally rapid in the presence or in the absence of added buffers;(d) the apparent substrate binding is weaker than predicted if steady-state Km values are assumed to represent substrate dissociation constants. The main conclusion concerning the catalytic mechanism of the enzyme is that the proton-transfer processes which limit turnover rates in the steady state are not directly involved in CO2-HCO-3 exchange. In addition, the results suggest that CO2-HCO-3 interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH- with CO2.
Mesh-terms: Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Trichomonas vaginalis :: pathogenicity; Keywords: co2-hco; anhydrase; carbonic anhydrase; human carbonic; carbonic; exchange; nuclear-magnetic-resonance; chemical equilibrium; equilibrium; h2o; exchange rate; equally rapid; catalyz; turnover rate; turnover;
Rose Mikulski, John F Domsic, George Ling, Chingkuang Tu, Arthur H Robbins, David N Silverman, Robert McKenna
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, United States.
The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1μs(-1), determined by exchange of (18)O between CO(2) and water measured by mass spectrometry. The apparent values of the pK(a) of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO(2) and bicarbonate.
Catalysis and pH control by membrane-associated carbonic anhydrase IX in MDA-MB-231 breast cancer cells.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using (18)O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO(2) species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Balendu Sankara Avvaru, Chae Un Kim, Katherine H Sippel, Sol M Gruner, Mavis Agbandje-McKenna, David N Silverman, Robert McKenna
Department of Biochemistry and Molecular Biology, Universityof Florida, Gainesville, Florida 32610, USA.
The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 A resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 A. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO(2) in the enzyme-substrate complex, a weak binding that enhances k(cat) for the conversion of CO(2) into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pK(a) of the zinc-bound water and to promote proton transfer in catalysis.
Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.
Center for Biophysical Modeling and Simulation and the Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
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.
13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s-1 at 20 degrees C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN-) was also probed by 13C NMR. A functional C-cluster is not required for 13CN- broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN- exchange appears to monitor only movement of CN- between solution and its binding to and release from CODH.
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.
S Fisher, Chingkuang Tu, Deepa Bhatt, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, Robert McKenna, David Silverman
Departments of Biochemistry and Molecular Biology, and Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida 32610.
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 mus-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.
University of Florida.
Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp64 about 8 A from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of about four-fold greater activation with the mutant containing Trp64 compared with Gly64, and the observation by X-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.
Department of Pharmacology and Therapeutics University of Florida, Gainesville, Florida 32610-0245, USA.
Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer
Other papers by authors:
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.
A mechanism model has been presented that can describe most known kinetic properties of carbonic anhydrase isoenzymes I, II, and III. The essential features of this model include: Nucleophilic attack of metal-bound OH- on CO2 to form metal-bound HCO-3. Formation of metal-bound OH- from metal-bound H2O. In isoenzyme II, and probably also in isoenzyme I, this reaction step involves an intramolecular transfer of H+ between the metal site and a titratable histidine residue via a number of hydrogen-bonded H2O molecules. In isoenzyme II, this step limits the maximal rate of catalysis. Also in isoenzyme III, the H2O-splitting step may be rate limiting, but since this isoenzyme has no titratable active-site histidine, H+ transfer may take place directly with components of the solvent. In isoenzymes I and II, rapid H+ transfer between active site and solution proceeds in a reaction between the titratable histidine residue and buffer molecules. The model can also rationalize a variety of observed inhibition patterns.
Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.
Department of Biochemistry, Umeå University, Umeå, S-90187, Sweden.
The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8%(Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.
The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae.
Department of Biochemistry, Umeå University, Sweden.
The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252-residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26-residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an alpha-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and II suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active-site residues are identical to those found in the high-activity human isozyme II. The bacterial enzyme has a high CO2 hydration activity with a k(cat) of 1.1 x 10(6) s(-1) and Km of 20 mM at pH 9 and 25 degrees C. The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pKa of 6.7 and a maximal value of the catalytic second-order rate constant, k(enz), of 130 M(-1) x s(-1).
Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme.
Department of Biochemistry, Umeå University, Sweden.
A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity. The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32,950 Da. By introducing two mutations, Arg117 --> His and Glu115 --> Gln, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1:1 zinc-protein complex. While unmodified CARP is catalytically inactive, the mutant catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide.
Crystallographic analysis of Thr-200-->His human carbonic anhydrase II and its complex with the substrate, HCO3-.
Department of Biochemistry, University of Umeå, Sweden.
A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO3- ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO3- is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 A resolution.
Importance of the conserved active-site residues Tyr7, Glu106 and Thr199 for the catalytic function of human carbonic anhydrase II.
Avdelningen för biokemi, Umeå Universitet, Sweden.
The catalytic mechanism of carbonic anhydrase includes the reaction of a zinc-bound hydroxide ion with the CO2 substrate. This hydroxide ion is part of a hydrogen-bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site-specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gln and Asp, and Tyr7 to Phe. The effects of these mutations on catalyzed CO2 hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO2 hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen-bonded network. The CO2 hydration activity (kcat as well as kcat/Km) and the esterase activity of the mutant with Ala199 are reduced about 100-fold. The affinity for the sulfonamide inhibitor, dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN-. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100-fold, while kcat for CO2 hydration has decreased by a factor of 1000. The parameter kcat/Km is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20-times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199.
Proton transfer in the catalytic mechanism of carbonic anhydrase. Effects of placing histidine residues at various positions in the active site of human isoenzyme II.
Avdelningen för Biokemi, Umeå Universitet, Sweden.
The maximal rate of CO2 hydration catalyzed by human carbonic anhydrase II (carbonate hydro-lyase, EC 184.108.40.206) is limited by proton transfer steps involving the acid-base function of His-64. To test whether or not the precise location of this proton transfer group is critical, histidine residues were placed in various positions in the active site of the enzyme. Thus, four double mutants were made, all with His-64 replaced by Ala-64, and with a histidine residue replacing Asn-62, Ala-65, Asn-67 or Thr-200. The results show that the mutants with His-62, His-67 and His-200, but not the mutant with His-65, yield significantly higher kcat values for CO2 hydration than the single mutant with Ala-64, indicating that His-62, His-67 and His-200 can contribute to proton transfer between the metal center and the reaction medium. However, the average proton transfer efficiency of these histidines is only about 5% of that of His-64 in the unmodified enzyme.
Modification of a metal ligand in carbonic anhydrase: crystal structure of His94-->Glu human isozyme II.
Department of Biochemistry, Umeå University, Sweden.
One of the zinc ligands in human carbonic anhydrase II, His94, has been replaced with glutamic acid by site-directed mutagenesis. The mutation leads to a less stable zinc binding site and to significant non-local perturbations of the protein structure. The crystals are composed of a mixture of holo- and apoenzyme, and the side chain of Glu94 has two conformations. In the holoenzyme, Glu94 coordinates to the metal ion and is hydrogen bonded to Gln92. In the apo form, Glu94 is hydrogen bonded to Asn67. The mutation has resulted in a 500-fold decrease of the catalyzed rate of CO2 hydration (kcat/Km).