Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.
The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D000644:Quaternary Ammonium Compounds; D011770:Pyruvate Kinase; D011188:Potassium