Turnover Is Rate-Limited by Deglycosylation for Micromonospora viridifaciens Sialidase-Catalyzed Hydrolyses: Conformational Implications for the Michaelis Complex
Turnover Is Rate-Limited by Deglycosylation for Micromonospora viridifaciens Sialidase-Catalyzed Hydrolyses: Conformational Implications for the Michaelis Complex
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DOI：
10.1021/ja109199p
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发表时间：
2011-03-09
影响因子：
15
通讯作者：
Bennet, Andrew J.
中科院分区：
文献类型：
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作者：
Chan, Jefferson;Lu, April;Bennet, Andrew J.
以7-（5-乙酰氨基-3,5-二脱氧-d -甘油- α -d -半乳糖-非2-乙酰氨基吡喃sylonic酸）-(2 - bbb6) β -d -半乳糖吡喃syloxy)-8-氟-4-甲基香豆素（1,Neu5Ac α 2,6ga1 β FMU）为核心结构，合成了7个同位素取代的唾液苷天然底物类似物，并用于探测M viridifaciens唾液酸酶转换的限制步骤。推导出的动力学同位素效应（KIEs）对k（cat）的影响分别为：环氧（V-18）、残基氧（V-18）、异头碳（V-13）、c3 -碳（V-13）、C3-R氘（V-D(R)）、C3-S氘（V-D(s)）和c3 -二氘((D)2(V))，分别为0.986 +/- 0.003、1.003 +/- 0.005、1.021 +/- 0.006、1.001 +/- 0.008、1.029 +/- 0.007、0.891 +/- 0.008和0.890 +/- 0.006。溶剂KIE （V-D2O）用于唾液酸酶催化水解！等于1.585 +/- 0.004。此外，测量了饱和条件下水解速率的线性质子库存，作为溶剂中氘分数n的函数。这些KIEs与酶促酪氨酸基-唾液皂苷中间体的速率决定切割相容。此外，仲氘KIEs与累积的Michaelis复合物一致，其中碳水化合物底物的盐酰环呈S-6(2)斜船构象。这些KIE测量也与通过爆炸过渡态发生的速率决定脱糖基化反应相一致，其中同步电荷脱糖基化发生在环氧原子上。最后，质子存量和溶剂KIE的大小与脱糖基化是一致的，脱糖基化涉及的是离去的酪氨酸残基的一般酸催化质子化，而不是一般的碱辅助亲核水的攻击。
A panel of seven isotopically substituted sialoside natural substrate analogues based on the core structure 7-(5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-non-2-ulopyranosylonic acid)-(2 -> 6)beta-D-galactopyranosyloxy)-8-fluoro-4-methylcoumarin (1, Neu5Ac alpha 2,6Ga1 beta FMU) have been synthesized and used to probe the rate-limiting step for turnover by the M viridifaciens sialidase. The derived kinetic isotope effects (KIEs) on k(cat), for the ring oxygen (V-18), leaving group oxygen (V-18), anomeric carbon (V-13), C3-carbon (V-13), C3-R deuterium (V-D(R)), C3-S deuterium (V-D(s)), and C3-dideuterium ((D)2(V)) are 0.986 +/- 0.003, 1.003 +/- 0.005, 1.021 +/- 0.006, 1.001 +/- 0.008, 1.029 +/- 0.007, 0.891 +/- 0.008, and 0.890 +/- 0.006, respectively. The solvent deuterium KIE (V-D2O) for the sialidase-catalyzed hydrolysis of! is 1.585 +/- 0.004. In addition, a linear proton inventory was measured for the rate of hydrolysis, under saturating condition, as a function of n, the fraction of deuterium in the solvent. These KIEs are compatible with rate-determining cleavage of the enzymatic tyrosinyl beta-sialoside intermediate. Moreover, the secondary deuterium KIEs are consistent with the accumulating Michaelis complex in which the sialosyl ring of the carbohydrate substrate is in a S-6(2) skew boat conformation. These KIE measurements are also consistent with the rate-determining deglycosylation reaction occurring via an exploded transition state in which synchronous charge delocalimtion is occurring onto the ring oxygen atom. Finally, the proton inventory and the magnitude of the solvent KIE are consistent with deglycosylation involving general acid-catalyzed protonation of the departing tyrosine residue rather than general base-assisted attack of the nucleophilic water.