Protein-carbohydrate interactions defining substrate specificity in Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases as dissected by mutational analysis.
The carbohydrate-binding site of Bacillus macerans 1,3-1, 4-beta-D-glucan 4-glucanohydrolase has been analyzed through a mutational analysis to probe the role of protein-carbohydrate interactions defining substrate specificity. Amino acid residues involved in substrate binding were proposed on the basis of a modeled enzyme-substrate complex [Hahn, M., Keitel, T., and Heinemann, U. (1995) Eur. J. Biochem. 232, 849-859]. The effects of the mutations at 15 selected residues on catalysis and binding were determined by steady-state kinetics using a series of chromogenic substrates of different degree of polymerization to assign the individual H-bond and hydrophobic contributions to individual subsites in the binding site cleft. The glucopyranose rings at subsites -III and -II are tightly bound by a number of H-bond interactions to Glu61, Asn24, Tyr92, and Asn180. From k(cat)/K(M) values, single H-bonds account for 1.8-2.2 kcal mol(-)(1) transition-state (TS) stabilization, and a charged H-bond contributes up to 3.5 kcal mol(-)(1). Glu61 forms a bidentated H-bond in subsites -III and -II, and provides up to 6.5 kcal mol(-)(1) TS stabilization. With a disaccharide substrate that fills subsites -I and -II, activation kinetics were observed for the wild-type and mutant enzymes except for mutations on Glu61, pointing to an important role of the bidentate interaction of Glu61 in two subsites. Whereas removal of the hydroxyl group of Tyr121, initially proposed to hydrogen-bond with the 2OH of Glcp-I, has essentially no effect (Y121F mutant), side-chain removal (Y121A mutant) gave a 100-fold reduction in k(cat)/K(M) and a 10-fold lower K(I) value with a competitive inhibitor. In subsite -IV, only a stacking interaction with Tyr22 (0.7 kcal mol(-)(1) TS stabilization) is observed.
Piotukh
K
K
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain.
Serra
V
V
Borriss
R
R
Planas
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D004252:DNA Mutational Analysis; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D047071:beta-Glucans
C030561:4-methylumbelliferyl glycosides; D004187:Disaccharides; D004791:Enzyme Inhibitors; D005936:Glucans; D006027:Glycosides; D010940:Plant Proteins; D047071:beta-Glucans; D006923:Hymecromone; C067858:beta-glucan, (1-3)(1-4)-; D006026:Glycoside Hydrolases; C028020:licheninase; C015968:lichenin