Elleby B, Chirica L C, Tu C, Zeppezauer M, Lindskog S
Department of Biochemistry, Umeå University, Sweden.
Eur J Biochem. 2001 Mar;268(6):1613-9.
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 x 10(6) s(-1) 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 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.
我们研究了淋病奈瑟菌碳酸酐酶（NGCA）的稳态和平衡动力学性质。定性地说，该酶表现出与研究充分的人类碳酸酐酶II（HCA II）相同的动力学行为。这反映在CO₂水合动力学参数的相似pH依赖性以及化学平衡时CO₂与水之间¹⁸O交换动力学的相似行为上。催化常数k(cat)的pH谱可描述为一条滴定曲线，在碱性pH下具有异常高的最大值1.7×10⁶ s⁻¹，pK(a)为7.2。在pH 9时，k(cat)以可饱和的方式依赖于缓冲剂，表明存在以缓冲剂为第二底物的乒乓机制。k(cat)/K(m)比值取决于两个电离，pK(a)值分别为6.4和8.2。然而，¹⁸O交换测定在k(cat)/K(m)的pH谱中仅鉴定出一个可电离基团，表观pK(a)为6.5。对该细菌酶的His66→Ala变体的动力学分析结果表明，NGCA中的His66与HCA II中的His64作为质子穿梭体具有相同的功能。突变体中的动力学缺陷可被某些缓冲剂（如咪唑和1,2 - 二甲基咪唑）部分克服。该细菌酶对抑制剂NCO⁻、SCN⁻和N₃⁻的K(i)值与HCA II相似，而CN⁻和磺酰胺乙氧唑胺对该细菌酶的抑制作用比对HCA II弱得多。Co(II)取代的NGCA与乙酰唑胺、NCO⁻、SCN⁻、CN⁻和N₃⁻的加合物的吸收光谱类似于用人Co(II) - 同工酶I和II获得的相应光谱。盐酸胍（GdnHCl）诱导变性的测量揭示了CO₂水合活性对还原剂三（2 - 羧乙基）膦（TCEP）的敏感性。然而，A₂₉₂/A₂₆₀比值不受TCEP存在的影响，并且在2.8 - 2.9 M GdnHCl处观察到结构转变。
