Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila
Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila
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DOI：
10.1021/bi9828876
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发表时间：
1999-10-05
期刊：
影响因子：
2.9
通讯作者：
Ferry, JG
中科院分区：
文献类型：
--
作者：
Alber, BE;Colangelo, CM;Ferry, JG
用扩展X射线吸收精细结构（EXAFS）表征了嗜热甲烷八叠球菌（Methanosarcina thermophila）中原型γ-碳酸酐酶的锌和钴形式，并使用稳态分光光度法和（18）O交换平衡法研究了其动力学。EXAFS结果表明，钴类质同象取代锌和金属协调三个组氨酸和两个或三个水分子。在生理pH值下，Zn-Cam或Go-Cam对CO2水合的效率（k（cat）/K-m）比HCO 3-脱水高几倍，这一结果与Cam在乙酸盐上生长期间的拟议生理功能一致。对于Zn-和Go-Cam，CO2水合的稳态参数k（cat）依赖于pH，pk（a）为6.5-6.8，而k（cat）/ K-m依赖于两种电离，pk值为6.7-6.9和8.2-8.4。O-18交换试验还在k（cat）/K-m的pH曲线中鉴定了两个可电离基团，表观pK值为6.0和8.1。的稳态参数k（猫）（CO2水合）是缓冲液依赖的饱和方式在pH值8.2，和动力学分析建议乒乓机制，其中缓冲液是第二基板。计算得到的分子间质子转移速率常数为3 × 10（7）M ~（-1）s ~（-1）。在饱和缓冲液浓度和pH 8.5，K（猫）是2.6倍，在水比在D2 O，这表明分子内质子转移步骤是至少部分的速率决定。在高pH（pH > 8）下，k（cat）/K不依赖于缓冲液，并且没有观察到溶剂氢同位素效应，这与氢氧化锌机制一致。因此，在高pH值的凸轮的催化机制似乎类似于人类CAII，尽管这两个不相关的酶的活性位点的显着结构差异。
The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)0 exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Go-Cam for CO2 hydration (k(cat)/K-m) was severalfold greater than HCO3- dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Go-Cam, he steady-state parameter k(cat) for CO2 hydration was pH-dependent with a pk(a) of 6.5-6.8, whereas k(cat)/ K-m was dependent on two ionizations with pk values of 6.7-6.9 and 8.2-8.4. The O-18 exchange assay also identified two ionizable groups in the pH profile of k(cat)/K-m with apparent pK, values of 6.0 and 8.1. The steady-state parameter k(cat) (CO2 hydration) is buffer-dependent in a saturable manner at pH 8.2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M-1 s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H2O than in D2O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K, is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.