Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) requires two divale nt cations for activity. One cation activates the enzyme through a direct i nteraction with the protein at site nl. The second cation, at site n(2), ac ts in the cation-nucleotide complex that serves as a substrate. The Co3+-(n (1))-PEPCK and Cr3+(n(1))-PEPCK complexes were used to examine the kinetic, mechanistic, and binding properties of the n(2) metal. EPR studies perform ed on the Co3+(n(1))-PEPCK-GTP complex yielded a stoichiometry of 1 mol of Mn2+ bound per mole of Co3+(n(1))-PEPCK-GTP with a K-D of 5 mu M. PRR studi es show a significant enhancement for the Co3+(n(1))-PEPCK-Mn2+(n(2))-GDP c omplex. A change in enhancement in the presence of PEP suggests that PEP in teracts with the second metal ion. The distance between Mn2+ at site n(2) o n PEPCK and the cis and trans protons and the P-31 of PEP are 7.0, 7.5, and 4.8 Angstrom, respectively, as measured by high-resolution NMR. PRR studie s of the Co3+(n(1))-PEPCK-Mn2+-(n(2))-GTP and Co3+(n(1))-PEPCK-Mn2+(n(2))-G DP complexes as a function of frequency (omega(I)) were used to estimate th e hydration number of the n(2) metal to be between 0.5 and 0.7. The metal-m etal distance for the M(n(1))-PEPCK-M(n(2))-GTP complex is approximately 8. 3 Angstrom, and the distance for the M(n(1))-PEPCK-M(n(2))-GDP complex is 9 .2 Angstrom. The change in the metal-metal distance suggests a conformation al change at the active site of PEPCK occurs during catalysis. The Co3+(n(1 ))-PEPCK complex was incubated with Co2+, GTP, and H2O2 to create a doubly labeled and inactive Co3+(n(1))-PEPCK-Co3+(n(2))-GTP complex. The Co3+(n(1) )-PEPCK-Co3+(n(2))-GTP complex was digested by LysC, and two cobalt-contain ing peptides were purified using RP-HPLC. Amino acid sequencing of the seco nd 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.