Tu C, Qian M, Earnhardt J N, Laipis P J, Silverman D N
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville 32610-0267, USA.
Biophys J. 1998 Jun;74(6):3182-9. doi: 10.1016/S0006-3495(98)78024-5.
We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.
我们在一系列人碳酸酐酶III（HCA III）的位点特异性突变体中，研究了谷氨酸64位和天冬氨酸64位作为锌结合氢氧化物质子供体的效率。通过质谱法测量酶中H2 18O的质子转移依赖性释放速率，确定了这种分子内质子转移（碳酸酐催化脱水过程中的一个步骤）的速率常数。代表这些速率常数的自由能图可以用马库斯速率理论拟合，得出质子转移的内在能垒ΔG0++ = 2.2 +/- 0.5千卡/摩尔，以及对反应自由能的功函数或热力学贡献wr = 10.8 +/- 0.1千卡/摩尔。这些值在大小上与描述HCA III突变体中从His64和His67到锌结合氢氧化物的分子内质子转移的马库斯参数非常相似。这一结果以及在CA III催化过程中Glu64和Asp64作为质子供体的等效效率，表明从这些位点进行质子转移缺乏特异性，这是通过不同结构的中间水链存在多个质子传导途径的间接证据。所有这些质子转移中热力学贡献或功函数的主导地位与这样一种观点一致，即在此类水链中氢键的形成和断裂是质子转运的限制因素。
