Pyruvate carboxylase: Nuclear magnetic resonance studies of the enzyme-manganese-oxalacetate and enzyme-manganese-pyruvate bridge complexes

Abstract

Examination of the effects of the bound manganese of pyruvate carboxylase purified from chicken liver on the longitudinal ( 1 T 1 ) and transverse ( 1 T 2 ) nuclear magnetic relaxation rates of the methylene protons of oxalacetate indicates that these effects are enhanced approximately 13-fold on 1 pT 1p and 1.1-fold on 1 pT 2p as compared with those observed for Mn 2+. The average manganese-methylene proton distance calculated from 1 pT 1p is consistent with the formation of a pyruvate carboxylase-manganese-oxalacetate bridge complex but the data do not permit an unequivocal assignment of the structure of this complex. The rate of exchange of oxalacetate into the coordination sphere of the bound manganese obtained from 1 pT 2p is several orders of magnitude faster than the maximal turnover number for HCO 3 − fixation on pyruvate. Formation of the pyruvate carboxylase (avidin) 4 complex decreases the rate of exchange of oxalacetate into the coordination sphere of the bound manganese. The first-order rate constant for formation of the pyruvate carboxylase-manganese-oxalacetate bridge complex from an ion pair at 25 ° is equal to the rate of exchange of water molecules on the bound manganese, suggesting that this bridge complex is formed by an S N 1 outer-sphere mechanism. Determination of the enthalpies of binding ( ΔH) for pyruvate and oxalacetate and enthalpies of activation ( ΔH ≠) for dissociation of these substrates from the bound manganese has permitted the construction of energy diagrams which describe the coordination process. These energy diagrams reveal that other processes, in addition to dissociation of a water molecule, contribute to the kinetic barrier, and may control the rate of complex formation at low temperatures. Addition of oxalacetate causes a decrease in the affinity of pyruvate carboxylase for oxalate which is approximately two orders of magnitude greater than that predicted if a simple competitive relationship existed for the interaction of these two ligands for the bound manganese. These observations have been interpreted to indicate that addition of oxalacetate induces pyruvate carboxylase to assume an altered conformation which exibits a decreased affinity for the inhibitor, oxalate.