Productive interactions between the two domains of pig heart CoA transferase during folding and assembly.
The enzyme CoA transferase from porcine heart (EC 2.8.3.5) is a homodimer; each subunit consists of two domains linked by a hydrophilic "hinge" region. We have prepared separate DNA segments encoding each of these domains. Incorporation of these two DNA segments within an operon or within two separate transcription units does not preclude the synthesis and assembly of CoA transferase in Escherichia coli. When the two domain fragments are produced and purified individually from separate cultures and subsequently mixed, enzyme activity accumulates to near wild-type levels only after a lengthy incubation. Each domain is more susceptible to aggregation than wild-type CoA transferase. Circular dichroism shows that, prior to mixing, the domains possess a different secondary structural profile compared to their counterparts in the native enzyme. However, mixing and incubation of the domains produces a complex with far-UV CD, fluorescence, and ultracentrifugation properties similar to those of wild-type CoA transferase. Finally, we show that the intact hydrophilic peptide which links the two domains is essential for the recovery of activity observed upon refolding of the denatured enzyme in vitro. These results indicate that the folding and assembly of pig heart CoA transferase require a productive interaction between its two domains, involving a substantial conformational rearrangement.
Rochet
J C
JC
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Oikawa
K
K
Hicks
L D
LD
Kay
C M
CM
Bridger
W A
WA
Wolodko
W T
WT
D000818:Animals; D002942:Circular Dichroism; D015256:Coenzyme A-Transferases / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine
D015256:Coenzyme A-Transferases; C021460:3-ketoacid CoA-transferase