Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium.
To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.
Kawai
Rie
R
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Igarashi
Kiyohiko
K
Kitaoka
Motomitsu
M
Ishii
Tadashi
T
Samejima
Masahiro
M
D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000378:metabolism; D043326:Glucan 1,3-beta-Glucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D009844:Oligosaccharides; D020075:Phanerochaete / Q000201:enzymology*; D013379:Substrate Specificity
D004187:Disaccharides; D009844:Oligosaccharides; C014637:laminaribiose; D043326:Glucan 1,3-beta-Glucosidase