The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase


This publication doesn't include Faculty of Medicine. It includes Faculty of Science. Official publication website can be found on


Year of publication 1999
Type Article in Periodical
Magazine / Source European Journal of Biochemistry
MU Faculty or unit

Faculty of Science

Field Genetics and molecular biology
Keywords beta-glucosidase; cysteine residues; disulfide bridge; structure-function relationships
Description The maize Zm-p60.1 gene encodes a beta-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 Km 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 alpha-helix packed against hydrophobic amino acid resides of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.
Related projects:

You are running an old browser version. We recommend updating your browser to its latest version.

More info