Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1
Zouhar, J., Vevodova, J., Marek, J., Damborsky, J., Su, X.-D., Brzobohaty, B.
PLANT PHYSIOLOGY 127: 973-985 (2001)
The maize Beta-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 A 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. Recently, these aglycone-active-site interactions were hypothesized to determine substrate specificity in inactive enzyme-substrate complexes of ZMGlu1, an allozyme of Zm-p60.1. Here we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased Km of all mutants confirmed the involvement of F193 in determining enzyme affinity towards substrates with an aromatic aglycone. Unexpectedly, a 30-fold decrease in kcat was found in F193I mutant compared to 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 co-determines 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. Unexpectedly, 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.