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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.

SUBMITTER: Roberts D

PROVIDER: S-EPMC1161565 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

Project description:1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.

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

Project description:1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.

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