NEUTRAL IMIDAZOLE IS THE ELECTROPHILE IN THE REACTION CATALYZED BY TRIOSEPHOSPHATE ISOMERASE - STRUCTURAL ORIGINS AND CATALYTIC IMPLICATIONS
NEUTRAL IMIDAZOLE IS THE ELECTROPHILE IN THE REACTION CATALYZED BY TRIOSEPHOSPHATE ISOMERASE - STRUCTURAL ORIGINS AND CATALYTIC IMPLICATIONS
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DOI：
10.1021/bi00242a020
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发表时间：
1991-07-16
期刊：
影响因子：
2.9
通讯作者：
KNOWLES, JR
中科院分区：
文献类型：
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作者：
LODI, PJ;KNOWLES, JR
为了阐明组氨酸 95 在磷酸三糖异构酶介导的催化反应中的作用，对野生型酶和突变异构酶进行了 C-13 和 N-15 NMR 滴定研究，其中单个剩余组氨酸（位于活性位点）在咪唑环中同位素富集。 N-15 NMR 已证明特别有用，可以明确证明组氨酸 95 的咪唑环在异构酶活性的整个 pH 范围内（pH 5 至 pH 9.9 之间）不带电荷。结果要求组氨酸 95 的第一个 pK(a) 低于 4.5。这种异常低的 pK(a) 排除了传统观点，即组氨酸 95 的带正电荷的咪唑鎓阳离子向底物羰基氧上的形成电荷提供质子。 “在反应中间体类似物磷酸乙二醇羟肟酸存在下，对酶进行的 N NMR 实验表明，组氨酸 95 的 N-ε-2 和结合的抑制剂之间存在强氢键。这些发现表明，在催化反应中，从磷酸二羟丙酮的 C-1 中夺取质子首先产生烯二醇中间体，该中间体与组氨酸 95 的中性咪唑侧链形成强氢键合。咪唑质子参与该氢键的烯二醇随后质子化烯二醇，O-1 上的羟基质子的瞬时形成产生了另一种烯二醇中间体，该中间体崩溃产生产物甘油醛 3-磷酸，当认识到烯二醇的 pK(a) 值和组氨酸-95 咪唑环的扰动的 pK(a)2 时，这一最初令人惊讶的序列更加合理。可能彼此相当接近，从而允许两个容易且快速的质子转移，从而使两个烯二醇相互转化，据我们所知，这是第一个报道的咪唑侧链参与酶催化反应的例子。组氨酸-95的咪唑环位于短α-螺旋的氨基末端，根据取代咪唑在溶液中的行为，该螺旋将降低第一个和第二个。第二个组氨酸 95 侧链的 pK(a) 值。
To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, C-13 and N-15 NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. N-15 NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pK(a) of histidine-95 is below 4.5. This abnormally low pK(a) rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. "N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N-epsilon-2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate. This initially surprising sequence is more reasonable when it is recognized that the pK(a) values of the enediol and the perturbed pK(a)2 of the imidazole ring of histidine-95 may be rather close to each other, allowing for two facile and rapid proton transfers that interconvert the two enediolates. To our knowledge, this is the first reported example of the participation of an imidazolate side chain in an enzyme-catalyzed reaction. The imidazole ring of histidine-95 lies at the amino terminus of a short a-helix that will, in accord with what is known from the behavior of substituted imidazoles in solution, lower both the first and the second pK(a) values of the side chain of histidine-95.