Presteady-state kinetics of Bacillus 1,3-1,4-beta-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate.
In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-beta-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s(-1), was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.
Abel
M
M
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
Planas
A
A
Christensen
U
U
D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008962:Models, Theoretical; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase