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

ABSTRACT: 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.

SUBMITTER: Roberts D

PROVIDER: S-EPMC1162037 | biostudies-other | 1980 Sep

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1161565 | biostudies-other

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.

| S-EPMC1162038 | biostudies-other

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

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Imino proton NMR analysis of HDV ribozymes: nested double pseudoknot structure and Mg2+ ion-binding site close to the catalytic core in solution.

Project description:Minimized trans-acting HDV ribozyme systems consisting of three (Rz-3) and two (Rz-2) RNA strands were prepared and their folding conformations were analyzed by NMR spectroscopy. The guanosine residues in one of the enzyme components of Rz-3 were labeled with 13C and 15N. Imino proton signals were assigned by analysis of NOESY and HSQC spectra. The results are consistent with the nested double pseudoknot model, which contains novel base pairs (P1.1), as observed in the crystal structure of a genomic HDV ribozyme. The NOE connectivities suggest an additional G:G pair at the bottom of P1.1 and at the top of P4. The effects of temperature and Mg2+ ions on base pairs for Rz-3 were examined. The temperature variation experiment on Rz-3 showed that P3 is the most stable and that P1.1 is as stable as P1 and P2. The imino proton signals of the G:U pair at the bottom of P1 and the top of P1.1, which are close to the cleavage site, showed the largest changes upon Mg2+ titration of Rz-3. The results suggest that the catalytic Mg2+ ion binds to the pocket formed by P1 and L3.

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Characterizing riboswitch function: identification of Mg2+ binding site in T box antiterminator RNA.

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| S-EPMC2526249 | biostudies-literature

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

Project description:K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding.

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Role of Tissue Transglutaminase Catalytic and Guanosine Triphosphate-Binding Domains in Renal Cell Carcinoma Progression.

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| S-EPMC7643270 | biostudies-literature

Phosphofructokinase from Plasmodium berghei. Influence of Mg2+, ATP and Mg2(+)-complexed ATP.

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| S-EPMC1131295 | biostudies-other

Effects of substrate conformational strain on binding kinetics of catalytic antibodies.

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| S-EPMC5042168 | biostudies-literature

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