Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.
Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.
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DOI：
10.1021/bi060748f
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发表时间：
2006-07
期刊：
影响因子：
2.9
通讯作者：
Agoston Jerga;M. Stanley;P. Tipton
中科院分区：
文献类型：
--
作者：
Agoston Jerga;M. Stanley;P. Tipton
在线性多糖藻酸盐的合成中，C5-甘露糖醛酸差向异构酶催化从β-D-甘露糖醛酸残基形成α-L-古洛糖醛酸残基。该反应需要从进行差向异构化的残基的 C5 中提取一个质子，然后在相反的面上重新质子化。进行快速混合化学猝灭实验以确定在铜绿假单胞菌酶催化的反应中夺取质子时形成的中间体的性质。淬灭样品的比色和 HPLC 分析表明，含有不饱和糖残基的短寡糖在差向异构反应中形成为瞬时中间体。这表明碳负离子通过糖基形成而稳定，同时伴随着进行差向异构化的残基和相邻残基之间的糖苷键的断裂。糖醛形成的时间依赖性表明催化循环中化学步骤的侧面是缓慢的步骤。溶剂同位素对 V 和 V/K 的影响是一致的，与化学不受速率限制的催化循环一致。检查了差向异构酶对于相邻残基的特异性，并确定该酶对与古洛糖醛酸相邻的甘露糖醛酸残基与与甘露糖醛酸相邻的甘露糖醛酸残基没有表现出偏差。质子提取和糖差向异构化是不可逆的。多糖中已经存在的现有古洛糖醛酸残基没有转化为甘露糖醛酸，也没有观察到溶剂氘掺入现有的甘露糖醛酸中。
C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa. Colorimetric and HPLC analysis of quenched samples indicated that shortened oligosaccharides containing an unsaturated sugar residue form as transient intermediates in the epimerization reaction. This suggests that the carbanion is stabilized by glycal formation, concomitant with cleavage of the glycosidic bond between the residue undergoing epimerization and the adjacent residue. The time dependence of glycal formation suggested that slow steps flank the chemical steps in the catalytic cycle. Solvent isotope effects on V and V/K were unity, consistent with a catalytic cycle in which chemistry is not rate-limiting. The specificity of the epimerase with regard to neighboring residues was examined, and it was determined that the enzyme showed no bias for mannuronate residues adjacent to guluronates versus those adjacent to mannuronates. Proton abstraction and sugar epimerization were irreversible. Existing guluronate residues already present in the polysaccharide were not converted to mannuronates, nor was incorporation of solvent deuterium into existing mannuronates observed.