Stubbs H J, Brasch D J, Emerson G W, Sullivan P A
Department of Chemistry, University of Otago, Dunedin, New Zealand.
Eur J Biochem. 1999 Aug;263(3):889-95. doi: 10.1046/j.1432-1327.1999.00581.x.
The exo-beta-1,3-glucanase of Candida albicans (Exg) has a marked specificity for beta-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl beta-glucoside, beta-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The kcat/Km ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substrate-binding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the kcat twofold while positions +3 and +4 lower the Km value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminari-oligosaccharides which specifically form beta-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzyme-binding site.
白色念珠菌的外-β-1,3-葡聚糖酶（Exg）对β-1,3-糖苷键具有显著的特异性，这是根据对硝基苯基β-葡萄糖苷、葡萄糖的β-连接二糖（昆布二糖、龙胆二糖和纤维二糖）、海带糖系列寡糖、海带多糖和短梗霉多糖的动力学常数判断得出的。从二糖到七糖的一系列海带糖寡糖的kcat/Km比值表明，Exg具有一个扩展的底物结合位点，该位点包含至少五个糖残基结合位点。在+2位（第三个糖残基）结合会使kcat增加两倍，而+3和+4位会进一步降低Km值，从而提高催化效率。Exg能与高浓度的海带糖寡糖催化高效的转糖基化反应，这些寡糖特异性地形成β-1,3键，产率高达50%。转糖基化速率与浓度有关，比与过量供体底物（如海带三糖和海带六糖）的水解反应快10倍以上。Exg的动力学以及预测的多达五个糖残基的底物结合位点与该酶结合位点最近的结构分析结果一致。
