Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
J Biol Chem. 2011 Oct 14;286(41):35601-35609. doi: 10.1074/jbc.M111.280362. Epub 2011 Aug 12.
Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp(230) was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of k(cat) upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site.
海藻糖合酶 (TreS) 催化麦芽糖和海藻糖的可逆转化，最近研究表明其主要功能是作为糖原前体动员海藻糖。因此，这种有趣的异构酶的机制既有学术意义，也有潜在的药理学意义。TreS 还可催化 α-芳基葡萄糖苷以及α-葡萄糖基氟化物的水解裂解，从而可以方便地进行连续测定。TreS 与 5-氟糖苷反应，导致形成共价糖基-酶中间产物，这与 TreS 属于保留糖苷水解酶家族 13 酶家族一致，因此可能遵循两步、双置换机制。该捕获的中间产物经蛋白酶消化后进行 LC-MS/MS 分析，从而鉴定出 Asp（230）为催化亲核试剂。通过证明 TreS 无法将同位素标记的外源性葡萄糖掺入麦芽糖或海藻糖中，从而证明了异构化反应是一个分子内过程，这与先前对其他 TreS 酶的研究一致。不存在二级氘动力学同位素效应，并且 k(cat) 与离去基团能力基本无关，这都指向了一个决定速率的构象变化，可能是酶活性位点的打开和关闭。
