Plant Physiol, November 2001, Vol. 127, pp. 973-985

Insights into the Functional Architecture of the Catalytic Center of a Maize -Glucosidase Zm-p60.1¹

Jan Zouhar,² Jitka Vévodová,² Jaromír Marek, Jií Damborský, Xiao-Dong Su, and Betislav Brzobohatý^*

Department of Functional Genomics and Proteomics (J.Z., J.V., J.M., B.B.) and National Center for Biomolecular Research (J.V., J.D.), Faculty of Science, Masaryk University, Kotláská 2, CZ-61137 Brno, Czech Republic; Institute of Biophysics of the Academy of Sciences of the Czech Republic, Královopolská 135, CZ-61265 Brno, Czech Republic (J.Z., B.B.); and Department of Molecular Biophysics, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden (X.-D.S.)

The maize (Zea mays) -glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-Å 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, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K_m of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k_cat was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.