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• W2048478260 abstract "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 × 10 6 s −1 at alkaline pH and a p K 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 p K 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 p K a 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 K i values for the inhibitors NCO − , SCN − and N 3 − 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 N 3 − 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." @default.
• W2048478260 created "2016-06-24" @default.
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• W2048478260 date "2001-03-15" @default.
• W2048478260 modified "2023-10-03" @default.
• W2048478260 title "Characterization of carbonic anhydrase from<i>Neisseria gonorrhoeae</i>" @default.
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• W2048478260 doi "https://doi.org/10.1046/j.1432-1327.2001.02031.x" @default.
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