title: Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis.
abstract: A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.
authors.affiliation: Department of Biochemistry, University of Queensland, Brisbane, Australia.
authors.forename: R J
authors.initials: RJ
authors.lastname: Diefenbach
authors.forename: J M
authors.initials: JM
authors.lastname: Candy
authors.forename: J S
authors.initials: JS
authors.lastname: Mattick
authors.forename: R G
authors.initials: RG
authors.lastname: Duggleby
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011767:Pyruvate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics*
chemical_list: D001224:Aspartic Acid; D014364:Tryptophan; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate