Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene.
Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor beta-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor beta-1,3 glucan, yielding a linear glucan containing a beta-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-441]. An apparent Km of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent Km of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H2O competing for reaction at the activated donor site, thus resulting in hydrolysis. However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for beta-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of beta-1,3 glucan into the previously existing cell wall structure.
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064-3500.
R C
RC
Goldman
P A
PA
Sullivan
D
D
Zakula
J O
JO
Capobianco
D002176:Candida albicans / Q000201:enzymology / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics* / Q000378:metabolism; D005936:Glucans / Q000378:metabolism*; D007700:Kinetics; D009844:Oligosaccharides / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D047071:beta-Glucans
D005936:Glucans; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D047071:beta-Glucans; C033363:beta-1,3-glucan; C080389:BGL2 protein, S cerevisiae; D004693:Glucan Endo-1,3-beta-D-Glucosidase