Subsite specificity of the active site of glucosyltransferases from Streptococcus sobrinus.
Oral bacterial glucosyltransferases use sucrose as a substrate in synthesis of either alpha-1,3 water-insoluble glucans (GTF-I) or alpha-1,6 water-soluble glucans (GTF-S). The binding specificity of the glucosyl and fructosyl subsites of the sucrose-binding site was examined to identify ligands that bind exclusively to each subsite. Such compounds can be used as reporter ligands to localize the subsite binding of any reversible or irreversible active site inhibitor. In examining potential subsite-specific ligands, binding affinity to GTF-I was consistently stronger than binding to GTF-S. Fructose was found to be a moderate GTF inhibitor, but free glucose, alpha-methylglucoside and glucose epimers were very weak inhibitors. In contrast, glucose transition-state analogues, D-glucano-1,5-lactone, 1-deoxynojirimycin (dNJ), and most N-alkyl derivatives of dNJ were moderate to strong inhibitors; in particular N-methyl-dNJ was found to be the strongest GTF inhibitor identified to date. Multiple inhibitor kinetic analysis established nonexclusive binding of fructose and dNJ at the respective subsites. Binding of fructose and N-alkyl-dNJ derivatives was, to a small degree, partially exclusive. Fructose and dNJ were used as reporter ligands to localize the subsite specificity of two test inhibitors: a reversible inhibitor, Zn2+, and an irreversible inhibitor, diethyl pyrocarbonate (DEP). Zn2+ paired with dNJ in multiple inhibitor kinetic analysis showed no competition between the inhibitors, while Zn2+ paired with fructose decreased ligand affinity 7-fold, establishing Zn2+ binding exclusively at the fructose subsite. Analogous experiments adapted to the irreversible inhibitor DEP indicated that it reacts at both subsites or induces a protein conformational change at one subsite that alters ligand binding at the adjacent subsite.
Devulapalle
K S
KS
Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles 90089-0641.
Mooser
G
G
D001665:Binding Sites; D002236:Carbohydrate Conformation; D005947:Glucose / Q000031:analogs & derivatives / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009005:Monosaccharides / Q000494:pharmacology*; D016986:Streptococcus sobrinus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009005:Monosaccharides; D005964:Glucosyltransferases; D005947:Glucose