title: Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase.
abstract: Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison 53706.
authors.forename: D M
authors.initials: DM
authors.lastname: Kiick
authors.forename: W W
authors.initials: WW
authors.lastname: Cleland
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003653:Decarboxylation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D010071:Oxaloacetates / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D013053:Spectrophotometry; D013223:Statistics as Topic
chemical_list: D008670:Metals; D010071:Oxaloacetates; D003035:Cobalt; D008345:Manganese; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium