کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2050607 | 1074175 | 2006 | 6 صفحه PDF | دانلود رایگان |
Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M−1 s−1 (pH 5, 30 °C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert α-d-glucose 1-phosphate into β-d-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from α-retaining in wild-type to inverting in D196A. Phosphorolysis of β-d-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as α-glucosynthase.
Journal: FEBS Letters - Volume 580, Issue 16, 10 July 2006, Pages 3905–3910