Abstract
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).
Highlights
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
Men the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy
The identical results which were measured in the presence of the binary complex for monomethylammonium ion and dimethylammonium ion demonstrate that they bind at the same site on the enzyme, 8.7 %, from Mnz+, forming an active conformation
Summary
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. The conformation of the ternary enzyme .Mn*+ .phosphoenolpyruvate complex about the bound MnZ+, as reflected by the enhancement values (cT) measured, differs depending upon the nature of the monovalent cation. The identical results which were measured in the presence of the binary complex for monomethylammonium ion and dimethylammonium ion demonstrate that they bind at the same site on the enzyme, 8.7 %, from Mnz+, forming an active conformation. In the formation of the ternary complex the l/T, value for monomethylammonium ion increases by a factor of 7 reflecting a 2.2-A movement closer to the Mn*+ and provides a necessary change within the catalytic site to form the active complex. The l/T, value for the dimethylammonium ion increases only by a factor of 2 and reflects a 1-A movement in the formation of the ternary complex This change is insufficient to form an active complex. This complex must be able to undergo a substrate-induced conformational change to obtain the activated complex which is required for catalytic activity
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