The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

The maize Zm‐p60.1 gene encodes a β‐glucosidase that can release active cytokinins from their storage forms, cytokinin‐ O ‐glucosides. Mature catalytically active Zm‐p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site‐directed mutagenesis, and subsequent heterologous expression in Escherichia coli . All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea‐ and glutathione‐induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology‐based three‐dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the K m values for C170A/S mutants were equal to that for the wild‐type enzyme, C170 cannot participate in enzyme–substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k ′ cat . C170 is located on a hydrophobic side of an α‐helix packed against hydrophobic amino‐acid residues of β‐strand 4, indicating participation of C170 in stabilization of a (β/α) 8 barrel structure in the enzyme. In C479A/D/R/S mutants, K m and k ′ cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.