Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES) do not catalyse the refolding of mitochondrial malate dehydrogenase.
In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.
Miller
A D
AD
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, U.K.
Maghlaoui
K
K
Albanese
G
G
Kleinjan
D A
DA
Smith
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D018835:Chaperonin 10; D018834:Chaperonin 60; D004926:Escherichia coli / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D017510:Protein Folding; D013552:Swine
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; D008274:Magnesium; D011188:Potassium