tailieunhanh - Báo cáo khoa học: The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a b-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of ab-glycosidase from Spodoptera frugiperda(EC ), were investi-gated through site-directed mutagenesis and enzyme kinetic experiments. | ễFEBS Journal The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a b-glycosidase Lucio M. F. Mendonca and Sandro R. Marana Departamento de Bioquimica Institute de Quimica Universidade de Sao Paulo Sao Paulo Brazil Keywords aglycone catalysis glycoside hydrolase specificity b-glycosidase Correspondence S. R. Marana Departamento de Bioquimica Instituto de Quimica Universidade de Sao Paulo CP 26077 Sao Paulo 05513-970 SP Brazil Fax 55 11 3815 5579 Tel 55 11 3091 3810 E-mail srmarana@ Received 1 February 2008 revised 4 March 2008 accepted 13 March 2008 doi The relative contributions to the specificity and catalysis of aglycone of residues E190 E194 K201 and M453 that form the aglycone-binding site of a b-glycosidase from Spodoptera frugiperda EC were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones up to the sixth methylene group and also the first glucose unit of oligosaccharidic aglycones whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties whereas K201 is more relevant for the binding of glucose units in spite of its favorable interaction with alkyl moieties. The three residues E190 E194 and K201 reduce the affinity for phenyl moieties. In addition M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190 E194 K201 and M453 similarly contribute to stabilize ES . Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone but their joint catalytic effects are .