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-Row experiments performed on Bacillu s 1,3-1,4-beta -glucanases are reported. The presteady state kinetics of th e binding of 4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucoside t o the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bo und substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase l eading to equilibration (for binding to E134A) or to steady state product f ormation (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-substra te 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 ra te-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change o f the productive ES complex into a form that binds a second substrate molec ule in a non-productive mode.