Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis

Russell J. Diefenbach, Judith M. Candy, John S. Mattick, Ronald G. Duggleby*

*Corresponding author for this work

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 μM for thiamin diphosphate and from 4.21 to 45 μM 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 of glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.

Original languageEnglish
Pages (from-to)95-98
Number of pages4
JournalFEBS Letters
Volume296
Issue number1
DOIs
Publication statusPublished - 13 Jan 1992
Externally publishedYes

Keywords

  • Pyruvate decarboxylase
  • Site-directed mutagenesis
  • Thiamin diphosphate binding
  • Zymomonas mobilis

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