Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases.
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases.
复制标题
β-半乳糖苷酶（大肠杆菌，lac Z）的结构-反应性关系。
作者：
Richard,JP;Huber,RE;Heo,C;Amyes,TL;Lin,S
测定了在E461G和E461Q β-半乳糖苷酶催化的半乳糖转移反应中，β-d-半乳糖酰基从半乳糖−酶中间体向阴离子亲核试剂转移的二级速率常数。半乳糖化E461G酶与叠氮离子反应的二级速率常数为4900 M-1s-1。相比之下，半乳糖基化野生型酶与叠氮化物离子没有检测到反应（Richard et al., 1995b）， E461G突变导致催化β-d-半乳糖基叠氮化物裂解的二级速率常数kcat/ km大幅降低，这是叠氮化物离子与半乳糖基-酶中间体反应的微观逆转。这些数据表明，E461G突变导致β-d-半乳糖酰基从β-半乳糖苷酶转移到叠氮离子的平衡常数增加了8000倍以上。我们认为，这种变化代表了半乳糖从天然酶转移到对葡萄糖-461羧酸基的热力学不利的质子化的耦合要求，但表达了叠氮离子对半乳糖从突变酶转移的完全化学亲和力，该突变酶在461位置缺乏这种可电离侧链。乙酸、丁酸和甲氧基乙酸离子与半乳糖化E461G酶的反应，以及乙酸与半乳糖化E461Q酶的反应，都得到了相应的β-半乳糖基衍生物和半乳糖，后者的形成代表了水与半乳糖化酶反应的形式催化。而甲酸离子与半乳糖化的E461G酶的反应只能得到半乳糖。这些结果表明羧酸阴离子可以取代Glu-461中被切除的丙酸侧链，为水与半乳糖酶中间体的反应提供一般的碱催化作用。在E461G酶催化的反应中，阴离子亲核试剂对共价半乳糖-酶中间体的相对反应活性与在水中观察到的稳定碳正离子的分配相似。这表明，用氢取代Glu-461的阴离子侧链，使酶稳定的氧羰基离子中间体与外部亲核试剂发生反应。
Second-order rate constants for transfer of the β-d-galactopyranosyl group from the galactosyl−enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q β-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constantkcat/Kmfor catalysis of cleavage of β-d-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl−enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the β-d-galactopyranosyl group from β-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding β-galactopyranosyl derivatives andd-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives onlyd-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl−enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl−enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.