Abstract
Abstract Pyruvate kinase, which has an absolute requirement for both a monovalent and a divalent cation for activity, can be activated by the monomethylammonium cation at a rate 0.5% that measured in the presence of K+. The kinetically determined dissociation constant for the monovalent activator in the presence of saturating substrates (K1 = 85 mm) agrees with that measured for K+ and the dissociation constant measured in the presence of saturating substrates and the divalent activator Mn2+, (K4 = 20.5 ± 3.3 mm) is somewhat larger than that for K+. The presence of carbonbound protons on the movovalent activator permits the investigation of the interaction of the monovalent activator with the enzyme-Mn2+ complexes by proton nuclear magnetic resonance techniques. The presence of the enzyme has no effect on the longitudinal (1/T1) and transverse (1/T2) relaxation rates of the carbon-bound protons. The formation of the enzyme-Mn2+ complex increases the relaxation rates due to the electron-nuclear dipolar interaction of the paramagnetic Mn2+ and the protons. In the presence of the substrate phosphoenolpyruvate, the relaxation rates are even further increased. A measure of the relaxation rates at two frequencies allows the evaluation of the correlation time for the electron-nuclear interaction, τc by several methods. No significant change in the correlation time is seen when the substrate complex is formed. The electron-nuclear distance can thus be evaluated from the correlation time, τc, and from the values measured for 1/T1. In the absence of substrate the protons of the methyl group are 8.3 ± 0.6 A from the enzyme-bound Mn2+. Upon formation of the pyruvate kinase-Mn2+-phosphoenolpyruvate complex, the protons move to 6.6 ± 0.2 A of the Mn2+. This result shows that the substrate affects the conformation of the active site by bringing the monovalent and divalent cation activators in closer proximity and that the monovalent activator can indeed bind in the active site of pyruvate kinase. The Mn2+-methyl proton distance measured is that predicted if the monovalent cation acts by binding P-enolpyruvate via the carboxyl group to form the active complex (Nowak, T., and Mildvan, A. S. (1972) Biochemistry 11, 2819). Monomethylammonium ion can thus serve as a useful probe in studying the interactions of monovalent cations with many biological systems with relative ease and should lead to a greater understanding of monovalent cation activation and function in biology.
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