Mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases: kinetics and pH studies with 4-methylumbelliferyl beta-D-glucan oligosaccharides.
The carbohydrate binding site of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase was probed with a series of synthetic 4-methylumbelliferyl beta-D-glucan oligosaccharides (1a-e). The title enzyme is a retaining endo-glycosidase that has an extended carbohydrate binding site composed of four glucopyranosyl binding subunits on the non-reducing end from the scissile glycosidic bond, plus two or three subsites on the reducing end. Subsites -II to -IV have a stabilizing effect on the enzyme-substrate transition state complex in the rate-determining step leading to a glycosyl-enzyme intermediate, with subsite -III having a larger effect (-3.5 kcal mol-1). Since KM values decrease from the mono- to the tetrasaccharide, part of the effect is due to ground stabilization of the Michaelis complex. On the other hand, the chromophoric trisaccharide 1c and the homologous nonchromogenic tetrasaccharide 2b, which locates a glucopyranosyl unit in subsite +I, have almost identical KM values, the difference in reactivity being a consequence of an 18-fold increase of kcat for 2b. Therefore, interactions between subsite +I and the substrate appear to be mainly used to lower the energy of the transition state in the glycosylation step, rather than in the stabilization of the Michaelis complex. Finally, the pH dependence of the kinetic parameters for the hydrolysis of 1c, and the pH-dependent enzyme inactivation by a water-soluble carbodiimide (EAC) suggest two essential groups with pKa values of 5.5 and 7.0 in the free enzyme. The latter value is shifted up to 1.5 pH units upon binding of substrate in the non-covalent enzyme-substrate complex.
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Laboratory of Biochemistry, Universitat Ramon Llull, Barcelona, Spain.
Planas
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D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D005936:Glucans / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D047071:beta-Glucans
D005936:Glucans; D005960:Glucosides; D009844:Oligosaccharides; D047071:beta-Glucans; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; C067858:beta-glucan, (1-3)(1-4)-; D006026:Glycoside Hydrolases; C028020:licheninase