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

Diefenbach R.J., Candy J.M., Mattick J.S. and Duggleby R.G. (1992) Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis. FEBS Letters, 296 1: 95-98. doi:10.1016/0014-5793(92)80411-9


Author Diefenbach R.J.
Candy J.M.
Mattick J.S.
Duggleby R.G.
Title Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis
Journal name FEBS Letters   Check publisher's open access policy
ISSN 0014-5793
Publication date 1992-01-13
Sub-type Article (original research)
DOI 10.1016/0014-5793(92)80411-9
Open Access Status
Volume 296
Issue 1
Start page 95
End page 98
Total pages 4
Subject 1303 Specialist Studies in Education
1304 Biophysics
1312 Molecular Biology
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.
Keyword Pyruvate decarboxylase
Site-directed mutagenesis
Thiamin diphosphate binding
Zymomonas mobilis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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Created: Tue, 23 Aug 2016, 11:09:34 EST by System User