Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase.
Site-directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg(137)-->Ala (approximately 60-fold) and Tyr(340)-->Ala (approximately 2500-fold) concerned phosphate dependent half-reactions whereas that in Asp(338)-->Asn (approximately 7000-fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with alpha-d-glucose-1-phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild-type, 19 and 2.0 for Arg(137)-->Ala, 950 and 0.17 for Tyr(340)-->Ala, and 0.05 and 180 for Asp(338)-->Asn, respectively. Asp(338) and Tyr(340) have a key role in differential binding of fructose and phosphate, respectively.
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Mario
M
Mueller
Bernd
B
Nidetzky
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D005632:Fructose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010710:Phosphates; D005632:Fructose; D001224:Aspartic Acid; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D000409:Alanine