Cecilia Forsman

Kinetics and Mechanism of Carbonic Anhydrase Isoenzymes

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.

Anion Inhibition of CO2 hydration catalyzed by human carbonic anhydrase II: Mechanistic implications

Five monovalent anions, I-, N3-, SCN-, NCO- and Au(CN)2-, were investigated as inhibitors of CO2 hydration catalyzed by human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1). Predominantly uncompetitive inhibition patterns were observed at pH near 9 in all cases. While Dixon plots of Km/V vs. inhibitor concentration were linear, all the investigated anions except NCO- gave nonlinear Dixon plots of 1/V vs. inhibitor concentration. The anion SCN- was also tested at pH 7.4 and 6.4. Essentially noncompetitive patterns of inhibition of CO2 hydration were obtained at both these pH values. These results are analyzed in terms of two rivaling mechanism models, a kinetic scheme originally proposed by Steiner et al. (Steiner, H., Jonsson, B.-H. and Lindskog, S. (1975) Eur. J. Biochem. 59, 253-259) and a rapid-equilibrium kinetic scheme proposed by Pocker and Deits (Pocker, Y. and Deits, T.L. (1982) J. Am. Chem. Soc. 104, 2424-2434). It is concluded that the observed steady-state inhibition patterns are compatible with both models, but that discriminatory data, strongly favouring the model of Steiner et al., are available in the literature.

Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II

To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a ‘proton shuttle group’ during catalysis of CO2‐HCO− 3 interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site‐directed mutagenesis. All these variants turned out to have high CO2 hydration activities. The k cat values at pH 8.8 and 25°C were only reduced by 1.5–3.5‐fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4‐nitrophenyl acetate. The pK a values for the activity‐controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major pK a shifted to 8.4

Structural Studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa: Inhibitor Complexes with Anions and Acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion — a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO2-HCO3 - interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli Identification of two cleavage sites

The chloroplast transit peptide (cTP) of pea carbonic anhydrase was shown to be processed at two different sites, giving protein subunits of two sizes. The cleavage sites were identified and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic residues, of the cTP. Properties of pea carbonic anhydrase produced in Escherichia coli show that folding, oligomerization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site I is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.

The inhibition of human carbonic anhydrase II by some organic compounds.

The inhibition of human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) by tetrazole, 1,2,4-triazole, 2-nitrophenol, and chloral hydrate has been investigated. These inhibitors, together with phenol which has been studied previously (Simonsson, I., Jonsson, B.-H. and Lindskog, S. (1982) Biochem. Biophys. Res. Commun. 108, 1406-1412), can be classified in three groups depending upon the kinetic patterns of inhibition of CO2 hydration at pH near 9. The first group, represented by tetrazole and 2-nitrophenol, yields predominantly uncompetitive inhibition under these conditions in analogy with simple, inorganic anions. The second group, represented by 1,2,4-triazole and chloral hydrate gives rise to essentially noncompetitive inhibition patterns, whereas phenol, representing the third group, is a competitive inhibitor of CO2 hydration. These diverse inhibition patterns are discussed in terms of the kinetic mechanism scheme originally proposed by Steiner et al. (Steiner, H., Jonsson, B.-H. and Lindskog, S. (1975) Eur. J. Biochem. 59, 253-259.

Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II.

The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9. The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9. However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations. All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions. These results are interpreted in terms of different pathways for a rate-limiting proton transfer. In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64. Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants. The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants. The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups. Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient. At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64. The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively.

Chloroplast import and sequential maturation of pea carbonic anhydrase: the roles of various parts of the transit peptide

Chloroplast pea carbonic anhydrase is synthesised in the cytosol with an unusually long bipartite N‐terminal extension of the mature sequence previously proposed to serve as a transit peptide. Studies of import into pea chloroplasts show that the N‐terminal 69 amino acids of the previously proposed transit peptide is sufficient for translocation and localisation to the stroma, while the acidic C‐terminal part does not seem to have any function in these processes. Processing of the in vitro imported precursors is shown to be at a new cleavage site located in the middle of the actual transit peptide. The results indicate that maturation occurs in more than one step. The time‐course does not seem to be dependent on the age of the chloroplast but on the age of the translocated precursor.

Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides

A leaf cDNA library from hybrid aspen, Populus tremula × tremuloides, was constructed. From this two different cDNA clones, denoted CA1a and CA1b, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the β-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants.

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I.

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).