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)

Chloroplast carbonic anhydrase from Pisurn sativurn has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase I1 made. Pea carbonic anhydrase was found to be dependent on a reducing agent in order to retain the catalytic activity. Oxidised, inactive, enzyme could be activated by the addition of a SH-agent.However, such activation gave only 60% of the activity of an enzyme kept in a reduced state all the time. The kinetics of CO, hydration show an increase in k,,, as well as in k,,,/K,,, with pH, but the pH profile does not follow a simple titration curve. The pH dependence is more complicated and it seems as if there are several titratable groups affecting the activity. At pH 9 we obtain a turnover number of 4x10' s-' and a k,,,/K,,, value of 1.8X10HM-' s-' with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent K, values with respect to the total buffer concentration are found between 52-185 mM at neutral and high pH. At low pH the situation is complex with deviations from Michaelis-Menten kinetics.Chloroplast carbonic anhydrase from higher plants have been reported to have primary structures that are completely different from the enzyme from animals. In addition, we find the circular dichroic spectrum of pea carbonic anhydrase to be well distinguished from that of human carbonic anhydrase 11. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase I1 in catalysing the hydration of CO, However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.Carbonic anhydrase (CA; carbonate hydro-lyase) is a zinc-containing enzyme catalysing the reversible hydration of CO,. It is ubiquitous and found in vertebrates, invertebrates, higher plants, algae and in some bacteria. According to the primary structures it seems as if CA has appeared twice during evolution and thus can be divided into two groups. The first group, which is the most studied, contains all so-far-known animal isozymes and the periplasmic CA from Chlarnydomonas reinhardtii. The second group includes chloroplast CA from higher plants together with CA found in two procaryotes. The amino acid sequences for the animal CAs are found to be highly similar with several conserved regions, including the three histidine ligands of the catalytically active zinc ion (Venta et al., 1987). A water molecule is found as the fourth ligand, giving an almost tetrahedral coordination geometry around the zinc ion. In mammals seven different isozymes are known, where most interest has been focused on human CA I and I1 (HCA I and HCA 11) and bovine CA 111. These are monomers with molecular masses around 29 kDa. Their crystal structures have been solved at 0.2-nm resolution or higher and the folding of the polypeptide chain was found to be very similar for all three K...

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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.

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