The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate

Abstract

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state.

The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site

“…In the presence of cyclic AMP, Mg2+-1,N6etheno-ATP binds only to the catalytic site and the allosteric transition of the R conformation into the T conformation induced by Mg2+-1,N6-etheno-ATP is blocked, so that the enzyme remains entirely in the R-type conformation stabilized by cyclic AMP (Wolfman et al, 1978; Roberts & Kellett, 1979). [Mg2'-1,N6-etheno-ATP1 (pM) Fig.…”

“…All materials and methods were as previously described (Roberts & Kellett, 1979). Static titrations of rabbit skeletal muscle phosphofructokinase were carried out with a stock solution of Mg2+-_,N6etheno-ATP containing enzyme at the same concentration as in the titration cell.…”

“…etheno-ATP. This is also an efficient substrate and an effective allosteric inhibitor of phosphofructokinase (Secrist et al, 1972;Roberts & Kellett, 1979) and provides a suitable spectral signal which allows its reaction with phosphofructokinase to be observed in a stopped-flow fluorimeter. When a reaction mixture of Mg2+-l,N6-etheno-ATP and phosphofructokinase is excited at 285 nm, the time-course of Mg2+-_,N6-etheno-ATP fluorescence enhancement caused by energy transfer from enzyme to nucleotide is biphasic.…”

“…The fast phase represents binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by binding of Mg2+-1,N6-etheno-ATP to the inhibitory site (Roberts & Kellett, 1979). The ability of the stopped-flow fluorimeter to distinguish between the consequences of binding to the catalytic and inhibitory sites permits the study not only of binding to each site independently, but also of their interaction.…”

Etheno-Substituted Nucleotides and Coenzymes: Fluorescence and Biological Activity

“…The slow phase results from the allosteric transition of the R conformation into the T conformation induced by binding of Mg2+-EATP to the inhibitory site. 238 The rate constants for the binding and dissociation of EATP have been determined for the separate conformations . 239 Preincubation of phosphofructokinase with cyclic AMP, a powerful activator of phosphofructokinase, abolishes the slow phase of EATP fluorescence enhancement by inducing the R conformation and blocking the inhibitory conformational change.238 In a partial structural mapping of rabbit muscle phosphofructokinase, the distance between the CAMP binding site and the most reactive sulfhydryl group on the enzyme is calculated as 28 * 6 8, by the method of resonance energy transfer,'" as described in the section on Affinity Labeling.…”