Spin-labelled phosphofructokinase and its interactions with ATP and metal-ATP complexes as studied by magnetic-resonance methods.

Abstract

Rabbit skeletal muscle phosphofructokinase has been specifically modified at its most reactive thiol group with the nitroxide spin label, 4‐(2‐iodoacetamido)‐2,2,6,6–tetramethyl piperidinooxyl. The specific activity and allosteric kinetics are only slightly altered in the modified enzyme. The electron spin resonance spectrum of the labelled enzyme at pH 7.5, in the absence of stabilising ligands, indicates that the label has moderate mobility, with a rotational correlation time, τR, of 3.3 ns, which is greater than the mobility of the smallest fully active oligomer of 340000 molecular weight (τR= 90 ns). The spectrum is sensitive to pH and to the binding of ATP and its magnesium and manganous complexes. The binding of the metal complexes produces an immobilisation of the label (τR= 4.7 ns). In addition to this, MnATP causes a 74% quenching of the signal height as a result of dipolar spin‐spin interaction. At pH 7.5, MnATP and ATP bind competitively to the labelled enzyme with dissociation constants of 540 μM and 1.3 mM respectively. The binding of MgATP is sigmoidal with [S]0.5= 400 μM and a Hill interaction coefficient of 2.0. A two‐state model is proposed to account for the electron spin resonance spectral changes, in which the cooperative interactions are due to metal‐ATP binding at catalytic sites, and the dipolar interactions are due to metal‐ATP binding at a separate regulatory site closer to the spin label. The longitudinal relaxation rate of water protons at 35 MHz is not detectably altered by the presence of the spin label on the enzyme. An analysis of enhancement data obtained in the presence of manganous ions and ATP indicates that the enhancement of the ternary complex, ɛt, is unchanged compared with the native enzyme, but that the dissociation constant for Mn ATP increases from 260 μM in the native enzyme to 480 μM in the labelled enzyme. The quenching of the electron spin resonance spectrum and the enhancement in the ternary complex of enzyme with MnATP have together been used to estimate the manganese‐nitroxide distance. The longitudinal and transverse relaxation rates of the H‐2, H‐8, and H‐1′ protons in MgATP bound to the spin‐labelled enzyme are increased in comparison with the native enzyme due to the dipolar spin‐spin interactions. This allows the proton‐nitroxide distances to be estimated. The distances calculated from the dipolar interactions have been used to obtain a self‐consistent three‐dimensional model of the regulatory site in relation to the spin label.