Ligand-induced conformational transitions and secondary structure composition of chicken liver pyruvate carboxylase.
Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002645:Chickens; D002942:Circular Dichroism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011766:Pyruvate Carboxylase; D013050:Spectrometry, Fluorescence
D001639:Bicarbonates; D008024:Ligands; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase; D008274:Magnesium; D011188:Potassium