|Title||Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Authors||Zhang, R, Pan, YT, He, S, Lam, M, Brayer, GD, Elbein, AD, Withers, SG|
|Journal||JOURNAL OF BIOLOGICAL CHEMISTRY|
|Date Published||OCT 14|
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 alpha-aryl glucosides as well as alpha-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.