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 CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s−1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa 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 Ki values for the inhibitors NCO−, SCN− and N3− 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 N3− 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 CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8-2.9 mGdnHCl was observed.

Nyckelord

carbonic anhydrase

inhibition

kinetics

mutagenesis

Neisseria gonorrhoeae

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