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 t he role of protein-carbohydrate interactions defining substrate specificity . Amino acid residues involved in substrate binding were proposed on the ba sis of a modeled enzyme-substrate complex [Hahn, M., Keitel, T., and Heinem ann, 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 degre e of polymerization to assign the individual H-bond and hydrophobic contrib utions to individual subsites in the binding site cleft. The glucopyranose rings at subsites -III and -II are tightly bound by a number of I-I-bond in teractions to Glu61, Asn24, Tyr92, and Asn180. From k(cat)/K-M values, sing le H-bonds account for 1.8-2.2 kcal mol(-1) transition-state (TS) stabiliza tion, and a charged I-I-bond contributes up to 3.5 kcal mol(-1). Glu61 form s 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 subsite s -I and -II, activation kinetics were observed for the wild-type and mutan t enzymes except for mutations on Glu61, pointing to an important role of t he bidentate interaction of Glu61 in two subsites. Whereas removal of the h ydroxyl group of Tyr121, initially proposed to hydrogen-bond with the 20H o f Glcp-I, has essentially no effect (Y121F mutant), side-chain removal (Y12 1A mutant) gave a 100-fold reduction in k(cat)/K-M and a IO-fold lower Kr v alue with a competitive inhibitor. In subsite -IV, only a stacking interact ion with Tyr22 (0.7 kcal mol(-1) TS stabilization) is observed.