4-Methyl-7-thioumbelliferyl-beta-D-cellobioside: a fluorescent, nonhydrolyzable substrate analogue for cellulases.
The kinetics of cellulose binding and hydrolysis by cellulases is not well understood except at steady-state conditions. For use in studies of cellulase pre-steady-state and steady-state kinetics, we have prepared 4-methyl-7-thioumbelliferyl-beta-D-cellobioside (MUS-CB), a ground-state nonhydrolyzable analogue of the fluorescent cellulase substrate 4-methylumbelliferyl-beta-D-cellobioside (MU-CB). MUS-CB is not hydrolyzed by the catalytic domain of cellulase E1 from Acidothermus cellulolyticus under conditions where this enzyme rapidly degrades MU-CB. Thermodynamic parameters describing the steady-state binding of MUS-CB to Thermobifida fusca cellulase Cel6A are similar to those for MU-CB, indicating that MUS-CB can be used in place of MU-CB to study binding events in the Cel6A active-site cleft. In the pre-steady-state, MUS-CB binds to Cel6A by a simple, one-step bimolecular association reaction. It is anticipated that similar thio-containing 4-methylumbelliferyl compounds will have applications in studies of other enzyme systems.
Barr
Brian K
BK
Department of Chemistry, Loyola College in Maryland, Baltimore, Maryland 21210-2699, USA. bbarr@loyola.edu
Holewinski
Ronald J
RJ
D001665:Binding Sites; D020134:Catalytic Domain; D002475:Cellobiose / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D002480:Cellulase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005960:Glucosides / Q000737:chemistry*; D006027:Glycosides / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011485:Protein Binding; D013816:Thermodynamics; D014468:Umbelliferones / Q000737:chemistry / Q000494:pharmacology*
C453491:4-methyl-7-thioumbelliferylcellobioside; D005456:Fluorescent Dyes; D005960:Glucosides; D006027:Glycosides; D014468:Umbelliferones; D002475:Cellobiose; C082831:methyl beta-cellobioside; D006867:Hydrolases; D002480:Cellulase