Insights into the functional architecture of the catalytic center of a maize beta-glucosidase Zm-p60.1

The maize (Zea mays) beta -glucosidase Zm-p60.1 has been implicated in regu lation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structur e of the wild-type enzyme was solved at 2.05-Angstrom resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F 200, and F461 located opposite W373 in a slot-like aglycone-binding site. T hese aglycone-active site interactions recently were hypothesized to determ ine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1 , an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysi s of F193I/Y/W mutants. The decreased K-m of all mutants confirmed the invo lvement of F193 in determining enzyme affinity toward substrates with an ar omatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was fo und in F193I mutant compared with the wild type. Kinetic analysis and compu ter modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also cod etermines catalytic rate by fixing the glucosidic bond in an orientation fa vorable for attack by the catalytic pair, E186 and E401. The catalytic pair , assigned initially by their location in the structure, was confirmed by k inetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assemb ly of a catalysis-competent homodimer, suggesting novel links between the a ctive site structure and dimer formation.