The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis.
Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (k(cat)=200s(-1)). Glucosylation and deglucosylation steps of D295N were affected uniformly, approximately 10(4.3)-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 10(5)-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the beta-glucosyl enzyme intermediate by > or = 23kJ/mol.
Mueller
Mario
M
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Nidetzky
Bernd
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed
D001426:Bacterial Proteins; D001224:Aspartic Acid; D005964:Glucosyltransferases; C025901:sucrose phosphorylase