The Mechanism of Aconitase Action

Abstract

Abstract Pig heart muscle aconitase has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis and sedimentation in the ultracentrifuge. The protein has an s20,w = 6.16, a molecular weight of 89,000, a D20,w value of 6.68 x 10-7 cm2 per sec, a frictional ratio, f/f0, of 1.08, and an isoelectric point of 9.1. The presence of 1.02 ± 0.11 moles of iron per 89,000 g of aconitase is revealed by atomic absorption spectroscopy. At 77°K, the electron spin resonance spectrum of aconitase has a signal at g = 4.1 arising from high spin iron(III). The iron(III) is thought to have a structural rather than a catalytic role since the electron spin resonance spectrum is unchanged upon addition of iron(II), cysteine, or citrate. The optical spectrum of aconitase shows a broad nonspecific absorption from 300 to 600 nm at 25°. Aconitase specifically requires iron(II) for activity and the time-dependent activation by iron(II) is fit by the consecutive combination of 2 iron(II) ions with the enzyme. The transverse relaxation rate, 1/T2, of aconitase-bound iron(III) decreases with increasing temperature and the longitudinal relaxation rate, 1/T1, increases with temperature below 22° and decreases with increasing temperature above 22°, yielding a correlation time τc = 1 x 10-11 sec. The inequality of the longitudinal and transverse relaxation rates indicates that protons are rapidly exchanging (g6 x 105 sec-1) between bulk solvent and the coordination sphere of the iron(III). The molar relaxivity, 1/T1p[Fe], for aconitase (5900 m-1 sec-1) at 22° indicates that aconitase-bound iron(III) is about half as effective as free iron (III) in relaxing the protons of water. From the Solomon-Bloembergen equation and assuming an iron to proton distance of 2.4 ± 0.1 A from crystallographic data, it is calculated that 3 ± 1 water molecules remain in the inner coordination sphere of aconitase-bound iron(III).