Inactivation of chicken liver pyruvate carboxylase by 1,10-phenanthroline.
Inactivation of chicken liver pyruvate carboxylase by the chelating agent 1,10-phenanthroline follows pseudo-first-order kinetics. The hyperbolic dependence of the apparent first-order rate constant on 1,10-phenanthroline concentration is consistent with a two-step inactivation mechanism, in which 1,10-phenanthroline binds firstly to the enzyme, and secondly to the enzyme-bound Mn(II) ion. Binding of 1,10-phenanthroline to pyruvate carboxylase results in complete loss of ATP/Pi exchange activity, but only a 61% decrease in pyruvate/oxaloacetate exchange activity. The rate of inactivation is greater at low enzyme concentrations, implying that binding of 1,10-phenanthroline to monomers and dimers is preferred relative to that of tetramers. Furthermore, in the presence of acetyl-CoA, which stabilizes the tetrameric structure, no dependence of inactivation on enzyme concentration is observed. As monitored by gel-permeation liquid chromatography, formation of the enzyme-Mn(II)-phenanthroline complex results in loss of the tetrameric structure of the enzyme. From atomic-absorption measurements, inactivation by 1,10-phenanthroline also causes some loss of Mn(II) from the enzyme. It is concluded that the Mn(II) atom does not participate directly in the reaction mechanism, but may play a structural role essential to the integrity of the enzyme's tetrameric structure.
Carver
J A
JA
Department of Biochemistry, University of Adelaide, South Australia.
Baldwin
G S
GS
Keech
D B
DB
Bais
R
R
Wallace
J C
JC
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000818:Animals; D002645:Chickens; D007502:Iron Chelating Agents / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D010618:Phenanthrolines / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D011766:Pyruvate Carboxylase / Q000037:antagonists & inhibitors*; D013054:Spectrophotometry, Atomic
D007502:Iron Chelating Agents; D008024:Ligands; D010618:Phenanthrolines; D010710:Phosphates; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase; C025205:1,10-phenanthroline