Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes.
The citrate synthases of the gram-negative bacteria, Escherichia coli and Acinetobacter anitratum, are allosterically inhibited by NADH. The kinetic properties, however, suggest that the equilibrium between active (R) and inactive (T) conformational states is shifted toward the T state in the E. coli enzyme. We have now manipulated the cloned genes for the two bacterial enzymes to produce two chimeric proteins, in which one folding domain of each subunit is derived from each enzyme. One chimera (the large domain from A. anitratum and the small domain from the E. coli enzyme) is designated CS ACI::eco; the other is called CS ECO::aci. Both chimeras are roughly as active as the wild type parents, but their Km values for both substrates are lower than those for the E. coli enzyme, and NADH inhibition is markedly sigmoid, while that for E. coli citrate synthases is hyperbolic. Curve-fitting to the allosteric equation suggests that these differences are the result of the destabilization of the T state in the chimeras. The ACI::eco chimera exists almost entirely as a hexamer, like the A. anitratum enzyme, while the ECO::aci chimera, like the E. coli synthase, forms three major bands on nondenaturing polyacrylamide gels, two of them hexamers of different net charge, and one a dimer. These findings indicate that subunit interactions leading to hexamer formation in allosteric citrate synthases of gram-negative bacteria involve mainly the large domains. The chimeras are also used to show that the NADH binding site of E. coli citrate synthase is located entirely in the large domain. Sensitivity of the chimeras to denaturation by urea, to which the A. anitratum enzyme is much more resistant than the E. coli enzyme, is determined by the large domains. Sensitivity to inactivation by subtilisin is intermediate between those shown by the E. coli (very sensitive) and A. anitratum (quite resistant) synthases. This result suggests that digestibility by subtilisin is determined by conformational factors as well as the amino acid sequences of the target regions.
Molgat
G F
GF
Department of Chemistry, University of Manitoba, Winnipeg, Canada.
Donald
L J
LJ
Duckworth
H W
HW
D000150:Acinetobacter / Q000201:enzymology; D000249:Adenosine Monophosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D009243:NAD; D000249:Adenosine Monophosphate; D014508:Urea; D002950:Citrate (si)-Synthase; D013381:Subtilisins