Reversible Dissociation and Unfolding of Pyruvate Decarboxylase from Zymomonas mobilis

1 September 1994

journal article
research article
Published by Wiley in European Journal of Biochemistry

Vol. 224 (2) , 651-661
https://doi.org/10.1111/j.1432-1033.1994.0651a.x

Abstract

The denaturation and renaturation process of pyruvate decarboxylase (PDC) from Zymomonas mobilis (ATCC 29191) has been investigated using guanidine hydrochloride and urea as denaturing agents. The quarternary structure of the homotetramer is strongly stabilized by the cofactors Mg2+ and thiamine diphosphate (TDP). The structural transitions were monitored by activity measurements, fluorescence spectroscopy, circular dichroism and gel-filtration chromatography. A three-step denaturation process, described as follows, is indicated by non-coincidental denaturation curves: (a) inactivation of the tetramer upon dissociation of cofactors (> 0.4 M guanidine hydrochloride, > 1 M urea); (b) dissociation of the tetramer into monomers (> 1 M guanidine hydrochloride, > 3 M urea); (c) complete unfolding of these (> 2.5 M guanidine hydrochloride, > 5 M urea). The refolding process initiated by rapid dilution of fully denatured protein in renaturation buffer involves the rapid reassociation of an inactive intermediate followed by the reconstitution of the active site.

Keywords

This publication has 38 references indexed in Scilit:

Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992
Kinetic mechanism of pyruvate decarboxylase Evidence for a specific protonation of the enzymic intermediate
FEBS Letters, 1992
Effects of substitution of aspartate‐440 and tryptophan‐487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis
FEBS Letters, 1992
An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC)
FEBS Letters, 1990
Dissociation and aggregation of d-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate
Biochemistry, 1990
Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation
Biochemistry, 1984
Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol
Biochemical and Biophysical Research Communications, 1977
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry, 1976
Allosteric properties of yeast pyruvate decarboxylase
FEBS Letters, 1970