Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase.
Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) requires two divalent cations for activity. One cation activates the enzyme through a direct interaction with the protein at site n(1). The second cation, at site n(2), acts in the cation-nucleotide complex that serves as a substrate. The Co(3+)(n(1))-PEPCK and Cr(3+)(n(1))-PEPCK complexes were used to examine the kinetic, mechanistic, and binding properties of the n(2) metal. EPR studies performed on the Co(3+)(n(1))-PEPCK-GTP complex yielded a stoichiometry of 1 mol of Mn(2+) bound per mole of Co(3+)(n(1))-PEPCK-GTP with a K(D) of 5 microM. PRR studies show a significant enhancement for the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complex. A change in enhancement in the presence of PEP suggests that PEP interacts with the second metal ion. The distance between Mn(2+) at site n(2) on PEPCK and the cis and trans protons and the (31)P of PEP are 7.0, 7.5, and 4.8 A, respectively, as measured by high-resolution NMR. PRR studies of the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GTP and Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complexes as a function of frequency (omega(I)) were used to estimate the hydration number of the n(2) metal to be between 0.5 and 0.7. The metal-metal distance for the M(n(1))-PEPCK-M(n(2))-GTP complex is approximately 8.3 A, and the distance for the M(n(1))-PEPCK-M(n(2))-GDP complex is 9.2 A. The change in the metal-metal distance suggests a conformational change at the active site of PEPCK occurs during catalysis. The Co(3+)(n(1))-PEPCK complex was incubated with Co(2+), GTP, and H(2)O(2) to create a doubly labeled and inactive Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex. The Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex was digested by LysC, and two cobalt-containing peptides were purified using RP-HPLC. Amino acid sequencing of the second cobalt-containing peptide points to the region of Tyr57-Lys76 of PEPCK. Asp66, Asp69, and Glu74 are all feasible ligands to the site n(2) metal.
Hlavaty
J J
JJ
Department of Chemistry and Biochemistry, Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Nowak
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002857:Chromium / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry* / Q000378:metabolism; D010759:Phosphorus Isotopes; D013050:Spectrometry, Fluorescence; D013696:Temperature
D008670:Metals; D010446:Peptide Fragments; D010759:Phosphorus Isotopes; D002857:Chromium; D003035:Cobalt; D008345:Manganese; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D008274:Magnesium