Ren X, Tu C, Laipis P J, Silverman D N
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610-0267, USA.
Biochemistry. 1995 Jul 4;34(26):8492-8. doi: 10.1021/bi00026a033.
The ability of a histidine residue at position 67 in human carbonic anhydrase III to transfer protons in the catalytic pathway for the hydration of CO2 was investigated for a series of site-specific mutants. Wild-type carbonic anhydrase III has an arginine at this position with the C alpha of residue 67 about 9.4 A from the zinc. The active-site cavity contains no other residues capable of facile proton transfer. Rate constants for proton transfer from His 67 to the zinc-bound hydroxide were determined from the rate constants for the exchange of 18O between CO2 and water measured by mass spectrometry. A range of values for the pKa of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to the zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1.3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding unfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work function) that probably represents an energy requirement for proper alignment of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier appears the same for His 67 or His 64.
针对一系列位点特异性突变体，研究了人碳酸酐酶III中67位组氨酸残基在CO2水合催化途径中转移质子的能力。野生型碳酸酐酶III在该位置有一个精氨酸，67位残基的Cα距离锌约9.4埃。活性位点腔中没有其他能够轻松转移质子的残基。通过质谱法测量的CO2与水之间18O交换的速率常数，确定了从His 67向锌结合的氢氧化物转移质子的速率常数。通过将与锌相邻的198位的苯丙氨酸替换为亮氨酸和天冬氨酸，获得了锌结合水的一系列pKa值。Marcus速率理论的应用表明，涉及His 67的分子内质子转移具有1.3±0.3千卡/摩尔的内在能垒，以及先前不利平衡的热力学功函数为10.9±0.1千卡/摩尔。我们之前表明，碳酸酐酶III中组氨酸64的质子转移可以用Marcus速率理论来描述[Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757 - 10762]。相比之下，His 67的质子转移必须克服一个更不利的先前平衡（更大的功函数），这可能代表了供体和受体基团以及中间氢键水正确排列的能量需求。一旦实现这种排列，His 67或His 64的内在能垒似乎相同。
