Presteady-state kinetics of Bacillus 1,3-1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate
| Autor: | Ulla Christensen, Antoni Planas, Mireia Abel |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
chemistry.chemical_classification
Conformational change Glycoside Hydrolases Chemistry Stereochemistry Hydrolysis Kinetics Substrate (chemistry) Bacillus Cell Biology Models Theoretical Biochemistry Recombinant Proteins Substrate Specificity Spectrometry Fluorescence Reaction rate constant Amino Acid Substitution Phase (matter) Steady state (chemistry) Trisaccharide Molecular Biology Research Article |
| Zdroj: | Biochemical Journal. 357:195 |
| ISSN: | 0264-6021 |
| DOI: | 10.1042/0264-6021:3570195 |
| Popis: | In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-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 100s−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. |
| Databáze: | OpenAIRE |
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