Johansson I M, Forsman C
Department of Biochemistry, University of Umeå, Sweden.
Eur J Biochem. 1994 Sep 15;224(3):901-7. doi: 10.1111/j.1432-1033.1994.00901.x.
Chloroplast carbonic anhydrase from Pisum sativum has been studied to elucidate the catalytic mechanism and to test if the mechanism proposed for human carbonic anhydrase II is also valid for pea carbonic anhydrase. The catalytic activity was found to depend on the chemical nature of the buffer. Barbital buffer gives the highest turnover number at infinite buffer concentration and the lowest Km value with respect to the buffer, while the kinetic parameters obtained in the imidazole-type buffer, 1-methylimidazole, do not differ from those obtained using the biological-type buffer Mops. The anion inhibition of CO2 hydration was investigated using SCN- at pH 6-9. The binding of the anion was found to be pH dependent with the strongest interaction at low pH. We obtained an uncompetitive inhibition pattern at high pH and noncompetitive inhibition patterns at pH 7 and low pH. The catalytic mechanism was further tested by measurements of the solvent hydrogen isotope effects on the kinetic parameters for CO2 hydration. The observed effects were comparatively small with a kcat value of approximately 2 irrespective of the pH. The effect on kcat/Km and on Km changes when going from high pH to pH 7 and low pH. At high pH, the solvent isotope effect in Km is at least 3, giving a value below 1 for kcat/Km, while at pH 7 and low pH the major effect is found in kcat/Km with values of 2.6 and 2.9. The dependence of the CO2-hydration activity on the buffer concentration is in agreement with a ping-pong mechanism with buffer acting as a second substrate. This is analogous to the behaviour of human carbonic anhydrase II. The inhibition patterns and the observed isotope effects at high pH can also be explained within the framework of the catalytic mechanism for human carbonic anhydrase II, with a rate-determining and buffer-dependent part. The results are consistent with a mechanism involving a proton transfer that contributes to rate limitation. However, the isotope effects found at pH 7 and low pH indicate that some part of the mechanism has changed. Moreover, we cannot decide whether the mechanism for pea carbonic anhydrase involves an internal proton-shuttle group, or if the buffer molecule acts in a direct proton transfer from the zinc-coordinated water.
对豌豆叶绿体碳酸酐酶进行了研究，以阐明其催化机制，并检验提出的人碳酸酐酶II的机制是否也适用于豌豆碳酸酐酶。发现催化活性取决于缓冲液的化学性质。巴比妥缓冲液在无限缓冲液浓度下具有最高的周转数，并且相对于缓冲液具有最低的Km值，而在咪唑型缓冲液1-甲基咪唑中获得的动力学参数与使用生物型缓冲液Mops获得的参数没有差异。在pH 6 - 9下使用SCN-研究了阴离子对CO2水合作用的抑制。发现阴离子的结合与pH有关，在低pH下相互作用最强。在高pH下我们获得了非竞争性抑制模式，在pH 7和低pH下获得了非竞争性抑制模式。通过测量溶剂氢同位素对CO2水合动力学参数的影响，进一步检验了催化机制。观察到的影响相对较小，无论pH如何，kcat值约为2。从高pH到pH 7和低pH时，对kcat/Km和Km的影响会发生变化。在高pH下，Km中的溶剂同位素效应至少为3，使得kcat/Km的值低于1，而在pH 7和低pH下，主要影响出现在kcat/Km中，值分别为2.6和2.9。CO2水合活性对缓冲液浓度的依赖性与乒乓机制一致，其中缓冲液作为第二底物。这与人碳酸酐酶II的行为类似。高pH下的抑制模式和观察到的同位素效应也可以在人碳酸酐酶II的催化机制框架内得到解释，其中存在一个限速且依赖于缓冲液的部分。结果与涉及质子转移导致速率限制的机制一致。然而，在pH 7和低pH下发现的同位素效应表明机制的某些部分发生了变化。此外，我们无法确定豌豆碳酸酐酶的机制是否涉及内部质子穿梭基团，或者缓冲液分子是否在从锌配位水进行直接质子转移中起作用。
