Earnhardt J N, Qian M, Tu C, Laipis P J, Silverman D N
Department of Biochemistry, University of Florida College of Medicine, Gainesville 32610-0267, USA.
Biochemistry. 1998 May 19;37(20):7649-55. doi: 10.1021/bi9729209.
The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis. In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s-1 at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer. Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in kcat for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of 18O between CO2 and water measured by mass spectrometry. Comparison of kcat in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis. Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover kcat of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V. These replacements caused relatively small changes in kcat/Km for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.
碳酸酐酶催化二氧化碳水合作用时，每一轮催化都需要质子从活性位点与锌结合的水分子转移到溶液中。在哺乳动物中最有效的碳酸酐酶同工酶II中，这种转移由质子穿梭残基His 64促进。小鼠碳酸酐酶V（mCA V）在类似位置有一个空间受限的酪氨酸；它不是一个有效的质子穿梭体，但这种同工酶催化二氧化碳水合作用仍能在pH > 9时达到最大周转数3×10⁵ s⁻¹。我们研究了截短形式的mCA V中质子转移的来源，并确定了几个位于活性位点腔口附近的碱性残基，包括Lys 91和Tyr 131，它们有助于质子转移。通过停流分光光度法测量二氧化碳水合作用的kcat以及通过质谱法测量二氧化碳与水之间的¹⁸O交换时，这些穿梭基团与锌结合的水分子之间的分子内质子转移速率被估计为速率决定步骤。比较Lys 91和Tyr 131催化以及相应双突变体的kcat，发现这些位点之间存在强烈的拮抗相互作用，表明在促进催化的质子转移步骤中存在协同行为。替换四个潜在的质子穿梭残基产生了一个多重突变体，其催化周转数kcat仅为野生型的10%，这表明mCA V中的主要质子穿梭体已被考虑在内。这些替换对水合作用的kcat/Km造成的变化相对较小，kcat/Km衡量的是在催化的一个与质子转移分开且不同的阶段中二氧化碳和碳酸氢根的相互转化；这些测量作为对照，表明质子穿梭体的替换没有引起影响锌反应性的结构变化。
