The Mechanism of Aconitase Action: III. DETECTION AND PROPERTIES OF ENZYME-METAL-SUBSTRATE AND ENZYME-METAL-INHIBITOR BRIDGE COMPLEXES WITH MANGANESE(II) AND IRON(II)

Abstract

Abstract A magnetic resonance study of the paramagnetic contribution to the longitudinal (1/T1) and the transverse (1/T2) relaxation rates of the carbon-bound protons of citrate and trans-aconitate has been made utilizing Mn(II), Fe(II), and aconitase. The width of the methylene resonances in the nuclear magnetic resonance (NMR) spectrum of citrate (an AB pattern) increases linearly with Mn(II) concentration from 0 to 200 µm due to a paramagnetic effect on 1/T2 of these protons. Aconitase enhances the effect of Mn(II) on the 1/T1 and 1/T2 relaxation rates of the methylene protons of citrate and enhances the effect of Fe(II) on both relaxation rates of the —CH2— and —CHD—protons of pro-R monodeuterocitrate. From the Solomon-Bloembergen equation for 1/T1, the Mn(II) to proton distance in the aconitase-manganese-citrate ternary complex is 4.3 ± 0.5 A, and the Fe(II) to proton distances in the aconitase-iron-monodeuterocitrate ternary complex are 3.8 ± 0.2 A (—CH2—) and 3.1 ± 0.2 A (—CHD—). The absolute and relative values of these distances are in good agreement with crystallographic distances in the binary Mn(II)-citrate chelate complex. Hyperfine coupling of the unpaired electrons of enzyme-bound Fe(II) with the protons of citrate is detected. The data are, therefore, consistent with direct coordination of the substrate, citrate, by the metal ion on aconitase. In contrast with citrate, the NMR spectrum of the inhibitor trans-aconitate shows a nonlinear dependence of the width of the coupled methylene and methine resonances on the concentration of Mn(II) below 30 µm due to chemical exchange spin decoupling (Frankel, L. S. (1969) J. Mol. Spectrosc. 29, 273). Above 30 µm Mn(II), the resonance widths increase linearly with Mn(II) due to a simple paramagnetic effect on 1/T2. A ternary aconitase-manganese-trans-aconitate bridge complex is detected since aconitase enhances the effect of Mn(II) on the 1/T2 relaxation rate of the protons of trans-aconitate. From 1/T1, Mn(II) to proton distances in this ternary complex are 4.8 ± 0.5 A (—CH2—) and 5.3 ± 0.6 A (—CH—). As shown by NMR, both citrate and trans-aconitate compete for the metal site on aconitase at concentrations consistent with their respective dissociation constants as determined kinetically. From 1/T2, the aconitase-Fe(II)-citrate complex forms and dissociates rapidly enough to participate in enzyme catalysis. Hence the metal bridge complexes detected by NMR may be equated with the enzymatically active substrate complex and the enzymatically inactive inhibitor complex observed kinetically. Three mechanisms for the role of Fe(II) in the catalysis, consistent with the data, the forward and reverse ferrous wheel mechanisms, and the Bailar twist mechanism, are discussed.