Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate.
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 degrees C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert alpha-D-glucose 1-phosphate into beta-D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from alpha-retaining in wild-type to inverting in D196A. Phosphorolysis of beta-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 alpha-glucosynthase.
Schwarz
Alexandra
A
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Nidetzky
Bernd
B
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D002236:Carbohydrate Conformation; D002384:Catalysis; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics*; D010710:Phosphates / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D050505:Mutant Proteins; D010710:Phosphates; D001224:Aspartic Acid; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D006026:Glycoside Hydrolases; D005947:Glucose; D000409:Alanine