Novel tools for the study of class I alpha-mannosidases: a chromogenic substrate and a substrate-analog inhibitor.
The use of chromogenic substrates for evaluation of class I alpha-mannosidase is described. 2('),4(')-Dinitrophenyl-alpha-D-mannopyranoside allows rapid and sensitive assays of enzymatic activities, e.g., of heterologously expressed alpha-1,2-mannosidase from Trichoderma reesei. Interaction constants of several ligands with alpha-mannosidases from class I and II could also be determined. Furthermore, novel types of inhibitors derived from D-lyxose are presented. Methyl-alpha-D-lyxopyranosyl-(1(')-->2)-alpha-D-mannopyranoside is a potent inhibitor of the alpha-1,2-mannosidase from T. reesei (K(i)=600 microM) and since it probably spans subsites -1/+1, this disaccharide could be valuable in crystallographic studies of class I alpha-mannosidases.
Laboratory for Biochemistry, Department of Biochemistry, Physiology, and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
T
T
Desmet
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W
Nerinckx
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I
Stals
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N
Callewaert
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R
Contreras
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Claeyssens
D001667:Binding, Competitive; D002863:Chromogenic Compounds / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D008361:Mannosidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology; D043323:alpha-Mannosidase
D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D008361:Mannosidases; D043323:alpha-Mannosidase