The Mechanism of Aconitase Action II. MAGNETIC RESONANCE STUDIES OF THE COMPLEXES

When a number of metal ions were tested as activators over a lOO-fold range of metal ion concentration (0.01 to 1.0 mM), aconitase was specifically activated by Fe(II). Manganese(I1) does not activate aconitase but binds to the enzyme, as shown indirectly by inhibition of the enzyme activation by Fe(I1) and directly by electron paramagnetic resonance. A titration of the enzyme with Mn(I1) may be fit most simply by assuming two “tight” binding sites for Mn(I1) with a dissociation constant, Kdissp of 47 f 3 PM and 5 to 7 “weak” binding sites (Kdiss = 620 f 80 PM). Interaction among these sites is indicated by the effect of bound Mn(I1) on the longitudinal relaxation rate (l/TIP) of water protons. Iron(I1) competes with Mn(II) for the tight binding sites, yielding a dissociation constant for aconitase-Fe(I1) of 16 =t 6 PM. The binary aconitase-Fe(I1) complex and the ternary aconitase-Fe(II)-citrate complex are paramagnetic, as determined by l/Tl, of water protons, but less so than unbound Fe(II), as determined by magnetic susceptibility. In the catalytically inactive Mn-aconitase complex, a 3-fold enhancement of the effect of Mn on l/TIP of water protons is observed. Citrate, isocitrate, and cis-aconitate halve this enhancement, suggesting the formation of ternary enzymemetal-substrate (E-&f-S) bridge complexes. The dissociation constants (K3) of these ternary Mn(I1) complexes agree with the respective Km values of citrate and isocitrate, but not of cis-aconitate using the Fe(I1) enzyme. Citrate in proportion to its concentration raises the K3 of E-manganeseisocitrate, consistent with competition of the two substrates for the same Mn(I1) site on the enzyme.