Reversible dissociation and unfolding of pyruvate decarboxylase from Zymomonas mobilis.
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.
Pohl
M
M
Institut für Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Germany.
Grötzinger
J
J
Wollmer
A
A
Kula
M R
MR
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017124:Conserved Sequence; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014508:Urea; D017355:Zymomonas / Q000201:enzymology*
D006146:Guanidines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014508:Urea; D011767:Pyruvate Decarboxylase; D019791:Guanidine