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@article{223837, author = {Rotrekl, Vladimír and Nejedlá, Eliška and Kučera, Igor and Abdallah, Fuad and Palme, Klaus and Brzobohatý, Břetislav}, article_number = {3}, keywords = {beta-glucosidase; cysteine residues; disulfide bridge; structure-function relationships}, language = {eng}, issn = {0014-2956}, journal = {European Journal of Biochemistry}, title = {The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase}, volume = {266}, year = {1999} }
TY - JOUR ID - 223837 AU - Rotrekl, Vladimír - Nejedlá, Eliška - Kučera, Igor - Abdallah, Fuad - Palme, Klaus - Brzobohatý, Břetislav PY - 1999 TI - The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase JF - European Journal of Biochemistry VL - 266 IS - 3 SP - 1056 EP - 1056 SN - 00142956 KW - beta-glucosidase KW - cysteine residues KW - disulfide bridge KW - structure-function relationships N2 - 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. ER -
ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME a Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. \textit{European Journal of Biochemistry}. 1999, roč.~266, č.~3, s.~1056-1065. ISSN~0014-2956.
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