Magnetic Resonance Studies of Manganese (II) Binding Sites of Pyruvate Kinase: TEMPERATURE EFFECTS AND FREQUENCY DEPENDENCE OF PROTON RELAXATION RATES OF WATER

Abstract

The stoichiometry and dissociation constant of the binary rabbit muscle pyruvate kinase-manganese(II) complex have been investigated in the temperature range 5–37° by monitoring the amount of Mn(II) free in solution utilizing electron paramagnetic resonance spectrometry. The results have been interpreted in terms of an equilibrium mixture of two conformational forms of the enzyme, one which has four binding sites for Mn(II) (active) and another which does not bind Mn(II) (inactive). The equilibrium is affected by monovalent ions; potassium, an activator of the pyruvate kinase reaction, stabilizes the active form as compared to the enzymatically inert tetramethylammonium ion. Longitudinal proton relaxation rates of water in solutions of pyruvate kinase and Mn(II) have been measured by pulsed nuclear magnetic resonance spectrometry as a function of temperature (5–37°) and frequency (8 to 60.0 MHz) in order to gain some insight into the mechanism of relaxation and to determine the number of protein ligands to the metal. The proton relaxation rates due to bound Mn(II), highly enhanced relative to the Mn(II) aquo-ion, have been analyzed according to the Solomon-Bloembergen-Morgan scheme. The rationale and method of analysis are presented in detail. Unlike the Mn(II) aquo-ion for which the relevant correlation time for the interaction between Mn(II) and the water protons is the rotational time and consequently frequency-independent, it has been found that the relevant correlation time for proton relaxation of water in the vicinity of bound Mn(II) is, at least in part, frequency-dependent. The frequency-dependent component is identified with the longitudinal electron spin relaxation time of the bound Mn(II) and the other component with the mean residence time of a water molecule in the first hydration sphere, i.e. the reciprocal of the water ligand exchange rate. From the results the number of water molecules coordinated to Mn(II) bound to pyruvate kinase has been estimated to be 3. It may be concluded that the enzyme probably provides three ligands for Mn(II) and thus the conformational changes previously detected by protein difference spectroscopy due to the binding of a divalent metal ion may be rationalized. Although the binding constant of the binary Mn(II)-enzyme complex varies upon substitution of tetramethylammonium for potassium ion, the enhancement of the proton relaxation rate of water for the binary complex is invariant with monovalent ion.