Mechanism-Based Inhibition and Stereochemistry of Glucosinolate Hydrolysis by Myrosinase

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• 作者：Sylvain Cottaz ; Bernard Henrissat ; and Hugues Driguez
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：December 3, 1996
• 年：1996
• 卷：35
• 期：48
• 页码：15256 - 15259
• 全文大小：279K
• 年卷期：v.35,no.48(December 3, 1996)
• ISSN：1520-4995

文摘

Myrosinase is a particular glucosidase which hydrolyzesa variety of plant 1-thio--D-glucosidesknown as the glucosinolates. This enzyme, which is the onlyglycosidase able to hydrolyze these naturallyoccurring thioglucosides, has been found previously to display strongsequence similarities with family1 O-glycosidases. Myrosinase therefore offers theopportunity to compare the mechanism of enzymaticcleavage of S- vs O-glycosidic bonds. Thestereochemistry of hydrolysis of sinigrin by Sinapisalbamyrosinase was followed by ¹H NMR and the enzyme was foundto operate with a mechanism retainingthe anomeric configuration at the cleavage point exactly like therelated O-glycosidases found in family1. Myrosinase was readily inactivated by2-deoxy-2-fluoroglucotropaeolin with inactivation kineticparameters of K_i = 0.9 mM andk_i = 0.083 min^-1.Reactivation kinetic parameters were determined inbuffer only, with k_react = 0.015h^-1 and t_1/2 = 46 h, and alsoin the presence of acceptors oftransglycosylation. No significant changes were observed in thepresence of methyl -D-glucoside, butwith azide anion the half-life of reactivation was found to be reducedto t_1/2 = 20 h. These resultssuggestthat myrosinase inhibition by 2-deoxy-2-fluoroglucotropaeolin occursvia the accumulation of a long-lifeglucosyl-enzyme intermediate and that the catalytic machinery of theenzyme is composed of only onecatalytic residue, a nucleophilic glutamate, while the acid catalystresidue found in the correspondingO-glycosidases is missing.