Koenig S H, Brown R D
Proc Natl Acad Sci U S A. 1972 Sep;69(9):2422-5. doi: 10.1073/pnas.69.9.2422.
Carbonic anhydrase, a metalloenzyme containing one zinc atom per protein molecule of molecular weight 30,000, catalyzes the interconversion of CO(2) and HCO(3) (-) in solution. The rate of catalysis, among the fastest known, is pH-dependent, with a pK(Enz) near neutral. Arguments are presented to show that: (i) only the high-pH form of the enzyme is active both for the hydration and dehydration reactions (ii) at high pH there is an H(2)O ligand on the metal (not an OH(-) as is often argued), and (iii) the substrate for the dehydration reaction is the neutral H(2)CO(3) molecule. The arguments are based on data in the literature on the nuclear relaxation rates of Cl(-) ions and water protons in solutions of carbonic anhydrase, on strict application of the principle of microscopic reversibility, and on kinetic considerations. It has been argued that H(2)CO(3) cannot be the substrate for the dehydration reaction because the observed CO(2) production rate is somewhat faster than the maximum rate at which H(2)CO(3) molecules can diffuse to the active site of the enzyme. However, current models that consider HCO(3) (-) as the substrate implicity require that protons diffuse to the enzyme at an even greater rate, well outside the limitations imposed by diffusion. We consider two mechanisms to obviate the diffusion limitation problem, and conjecture that at high substrate concentration, H(2)CO(3) reaches the active site by collision with the enzyme molecule, and subsequent surface diffusion to the active site. At lower substrate concentrations, corresponding to [HCO(3) (-)] <1 mM, generation of H(2)CO(3) molecules near the enzyme by the recombination reaction H(+) + HCO(3) (-) --> H(2)CO(3) can supply an adequate flux of substrate to the active site.
碳酸酐酶是一种金属酶，每个分子量为30,000的蛋白质分子含有一个锌原子，它催化溶液中CO₂和HCO₃⁻的相互转化。催化速率是已知最快的速率之一，它依赖于pH值，其酶的pK值接近中性。本文提出的观点表明：（i）只有高pH形式的酶对水合和脱水反应均具有活性；（ii）在高pH值下，金属上存在一个H₂O配体（而非如通常所认为的OH⁻）；（iii）脱水反应的底物是中性的H₂CO₃分子。这些观点基于文献中关于碳酸酐酶溶液中Cl⁻离子和水质子的核弛豫速率的数据、微观可逆性原理的严格应用以及动力学考虑。有人认为H₂CO₃不可能是脱水反应的底物，因为观察到的CO₂产生速率比H₂CO₃分子扩散到酶活性位点的最大速率要快一些。然而，目前将HCO₃⁻视为底物的模型隐含地要求质子以甚至更快的速率扩散到酶，这远远超出了扩散所施加的限制。我们考虑了两种机制来消除扩散限制问题，并推测在高底物浓度下，H₂CO₃通过与酶分子碰撞到达活性位点，随后通过表面扩散到活性位点。在较低底物浓度下，对应于[HCO₃⁻]<1 mM，通过H⁺ + HCO₃⁻→H₂CO₃的重组反应在酶附近产生H₂CO₃分子可以为活性位点提供足够的底物通量。
