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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 25 314 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 a-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and I1 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 11. The bacterial enzyme has a high CO, hydration activity with a k,,, of 1.1 . lo6 s-' and K,, of 20 mM at pH 9 and 25°C.The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pK, of 6.7 and a maximal value of the catalytic second-order rate constant, k,,,,,

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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 Â 10 6 s 21 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 His663Ala 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 2 , SCN 2 and N 3 2 as HCA II, while CN 2 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 2 , SCN 2 , CN 2 and N 3 2 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.