Mutation of essential catalytic residues in pig citrate synthase.
Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375. The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity. Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum. No activity was detected for Asn375 or Gln375. The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity. The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs. A mechanism is proposed that electrostatically links His274 and Asp375.
Pre-Clinical Science Unit, Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
G M
GM
Alter
J P
JP
Casazza
W
W
Zhi
P
P
Nemeth
P A
PA
Srere
C T
CT
Evans
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000939:Epitopes; D004926:Escherichia coli / Q000276:immunology; D006639:Histidine / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D002950:Citrate (si)-Synthase