Wildberger Patricia, Luley-Goedl Christiane, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
FEBS Lett. 2011 Feb 4;585(3):499-504. doi: 10.1016/j.febslet.2010.12.041. Epub 2011 Jan 8.
Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe(52) replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (≥3.5kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild-type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe(52). In reverse reaction where fructose becomes glucocylated, "error hydrolysis" was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe(52) facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation-π stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.
通过自由能分布分析对肠系膜明串珠菌蔗糖磷酸化酶中活性位点苯丙氨酸（Phe52）被丙氨酸（F52A）或天冬酰胺（F52N）取代的突变体进行了表征，以研究催化蔗糖向磷酸转移葡萄糖基的过程。尽管与野生型相比，两个突变体中酶糖基化和去糖基化过渡态均有较大程度的不稳定（≥3.5千卡/摩尔），但苯丙氨酸（Phe52）的取代对糖基化酶中间体的相对稳定性影响较弱。在果糖发生糖基化的逆反应中，“错误水解”是F52A和F52N共价中间体分解的主要途径。因此，有人提出，苯丙氨酸（Phe52）通过以下方式促进磷酸化酶反应：（1）将转移的葡萄糖基部分定位在催化亚位点；（2）对共价酶中间体两侧的氧鎓离子样过渡态进行强阳离子-π稳定作用。
