Contribution of W229 to the transglycosylation activity of 4-alpha-glucanotransferase from Pyrococcus furiosus.
A W229H mutant of 4-alpha-glucanotransferase (4-alpha-GTase) from Pyrococcus furiosus was constructed and its catalytic properties were studied to investigate the role of W229 in the catalytic specificities of the enzyme. Various activities and kinetic parameters were determined for the wild-type and W229H mutant enzymes. The transglycosylation factor and transglycosylation activity of the mutant enzyme markedly decreased, but its hydrolysis activity was scarcely affected. It was discovered that the k(cat)/K(m) value of transglycosylation activity significantly decreased to about 15% of that of the wild type, while k(cat)/K(m) value of hydrolysis activity changed little for the mutant enzyme. The hydrophobicity of W229 was thought to be critical to the transglycosylation activity of the enzyme based on the enzyme's modeled tertiary structures.
Tang
Shuang-Yan
SY
Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Yang
Sung-Jae
SJ
Cha
Hyunju
H
Woo
Eui-Jeon
EJ
Park
Cheonseok
C
Park
Kwan-Hwa
KH
D000595:Amino Acid Sequence; D002384:Catalysis; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D006004:Glycogen Debranching Enzyme System; D014364:Tryptophan; C020569:4 alpha-glucanotransferase