The maize (Zea mays) 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. 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.

• MeSH
• beta-glukosidasa chemie   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kukuřice setá chemie   enzymologie   MeSH
• kvarterní struktura proteinů MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• molekulární struktura MeSH
• mutace MeSH
• nitrofenylgalaktosidy chemie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 4-nitrophenylgalactoside MeSH Prohlížeč
• beta-glukosidasa MeSH
• cytokinin-beta-glucosidase MeSH Prohlížeč
• nitrofenylgalaktosidy MeSH