Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli.
A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.
Evans
C T
CT
V.A. Medical Center, Dallas, Texas.
Owens
D D
DD
Slaughter
C A
CA
Srere
P A
PA
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010071:Oxaloacetates / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010446:Peptide Fragments; D010957:Plasmids; D013329:Structure-Activity Relationship; D013552:Swine
D000596:Amino Acids; D046911:Macromolecular Substances; D010071:Oxaloacetates; D010446:Peptide Fragments; C003712:trimethyllysine; D000105:Acetyl Coenzyme A; D004247:DNA; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine