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.