STEREOSPECIFICITY OF THE METAL ADENOSINE 5'-TRIPHOSPHATE COMPLEX IN REACTIONS OF MUSCLE PYRUVATE-KINASE

Rabbit muscle pyruvate kinase in the presence of mono- and divalent cations catalyzes the phosphorylation of glycolate by CrIIIATP. The product complex CrADP-glycolate-P is released slowly from the enzyme, and generally only a single turnover is observed for these reactions. The A β,γ-bidentate isomer and the four α,βγ-tridentate isomers are all inactive as substrates in this reaction. Only the Δ isomer of β,γ-bidentate CrATP is active in the phosphoryl transfer reaction and is the most active isomer in the enolization of pyruvate catalyzed by pyruvate kinase. In contrast to the phosphoryl transfer reaction, all of the bidentate and tridentate CrATP isomers are effective in promoting the pyruvate kinase catalyzed enolization of pyruvate. The Kmax of the most active isomer Δβ-γ-bidentate CrATP (Mn2+ and K+; pH 5.7; 21°C) is 6.0 μumol of pyruvate enolized per min mg of protein, and the Km is 100 μM. In comparison the Kmax of the A bidentate isomer under the same conditions is 3-fold lower and the Km is 1.7-fold greater. The four tridentate isomers of CrATP do not differ greatly in their activity but are all less active than the bidentate isomers by more than 1 order of magnitude. The most active tridentate isomer at pH 5.7 has a Vmax of 0.26μmol of pyruvate enolized per min mg of protein and a km of 600μM. Pyruvate kinase also requires an enzyme-bound divalent cation for activity. The order of activation by various enzyme-bound divalent cations in the phosphorylation of glycolate by CrATP is the same as it is in the CrATP-stimulated enolization of pyruvate: Mn(II) > Co(II), Zn(II)> Mg(II) >> Ca(II) ~ 0. The paramagnetic effects of the bidentate CrATP isomers on the relaxation rate of water protons are significantly lower than those of the tridentate isomers. Analysis of the temperature dependence of these effects indicates 1 order of magnitude slower escape of protons from the coordination sphere of bidentate CrATP (1.1 X 106 s_I) than that from tridentate CrATP, suggesting an intramolecular hydrogen-bond interaction in the bidentate isomers but not in the tridentate isomers. © 1979, American Chemical Society. All rights reserved.