Interaction of a spin-labeled analog of acetyl coenzyme A with citrate synthase. Paramagnetic resonance and proton relaxation rate studies of binary and ternary complexes

Weidman, Stuart W.; Drysdale, George R.; Mildvan, Albert S.

doi:10.1021/bi00734a006

Cited by 52 publications

(36 citation statements)

References 35 publications

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“…Carboxylic acids binding more strongly to the oxaloacetate site, however, will inhibit enzyme activity mainly through the latter interaction. This explains why the binding of oxoglutarate and trans-aconitate appears to be strictly competitive in the present kinetic experiments, while Weidman et al were able to detect binding to the acetyl-CoA site in their spectrometric experiments [20].…”

Section: Discussionmentioning

confidence: 51%

“…Such a catalytically adequate binding of the acetyl group in acetyl-CoA does not appear to take place at the level of binarycomplex formation, however. Association constants for the binding of CoASH and various acyl-CoA derivatives to free enzyme have been found to be of closely similar magnitude (about 10 mM-' [20]), indicating that binary-complex formation involves mainly interactions between enzyme and the CoA moiety of the ligands. This binding of the CoA portion, further, seems to be unaffected by the presence of oxaloacetate at the active center of the enzyme; oxaloacetate binds non-cooperatively with CoASH fragments such as ATP 161, as well as with acyl-CoA derivatives that do not function as substrates for the enzyme [5,6,20].…”

Section: Discussionmentioning

confidence: 92%

“…Association constants for the binding of CoASH and various acyl-CoA derivatives to free enzyme have been found to be of closely similar magnitude (about 10 mM-' [20]), indicating that binary-complex formation involves mainly interactions between enzyme and the CoA moiety of the ligands. This binding of the CoA portion, further, seems to be unaffected by the presence of oxaloacetate at the active center of the enzyme; oxaloacetate binds non-cooperatively with CoASH fragments such as ATP 161, as well as with acyl-CoA derivatives that do not function as substrates for the enzyme [5,6,20]. On the other hand, the affinity of citrate synthase for acetyl-CoA (and for the alternative substrate propionyl-CoA) increases at least 20-fold in the presence of oxaloacetate [5,6], which indicates that the acyl group contributes most significantly to binding in the ternary enzyme-substrate complexes.…”

Section: Discussionmentioning

confidence: 92%

“…Competition between the carboxylic acids and acetyl-CoA for the acetyl subsite, further, seems most unlikely in view of the evidence indicating that the acetyl group does not contribute significantly to the binding of acetyl-CoA in the binary complex. Similarly, displacement of the spin-labelled ,acetyl-CoA analogue in the experiments of Weidman et al [20] would appear to require displacement of the CoA moiety rather than displacement of the mobile (i.e. unbound) acyl group.…”

Section: Discussionmentioning

confidence: 93%

“…Direct evidence that the acyl portion of acyl-CoA derivatives does not participate in binding of the ligands to free enzyme has been provided by Weidman et al [20]. Using electron paramagnetic resonance techniques, these authors showed that the acyl group of spin-labelled acetyl-CoA analogues retains a high degree of mobility in the binary enzymic complex.…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of Pig‐Heart Citrate Synthase by Carboxylic Acids Structurally Related to Oxaloacetate

Johansson¹,

Petterson²

1979

European Journal of Biochemistry

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1. The kinetics of inhibition of citrate synthase by a number of dicarboxylic and tricarboxylic acids have been examined. Inhibition parameters providing information about equilibrium constants for ligand-binding to free enzyme and to the binary enzyme . oxaloacetate complex were determined under Michaelis-Menten conditions. Ligand-binding to the binary enzyme . acetyl-CoA complex was studied by a new kinetic approach, based upon an examination of the effect of inhibitors on the second-degree rate behaviour of the enzyme.2. Carboxylic acids may bind to the acetyl-CoA site (as anions), as well as to the oxaloacetate site (as oxaloacetate analogues). Depending on the relative strength of these interactions, inhibition competitive with either or both of the substrates is observed.3. Kinetically estimated equilibrium constants for the binding of inhibitors to different enzyme species in the catalytic mechanism are reported. The results obtained show that carboxylic acids, in general, bind non-cooperatively with the substrates.4. The affinity of citrate synthase for acetyl-CoA increases about 10-fold on the binding of D-malate. The mechanistic significance of this and previously reported heterotropic cooperativity effects in the citrate synthase system is discussed. It is suggested that the binding of oxaloacetate and certain carboxylic acids containing a D-malate substructure induces a conformational change of the enzyme. This conformational change serves the purpose of creating a binding site for proper accomodation of the acyl moiety of acetyl-CoA at the catalytic center of the enzyme.Citrate synthase catalyzes the condensation reaction between acetyl-CoA and oxaloacetate in the citric acid cycle. The enzyme operates by a basically random ternary-complex mechanism (Scheme 1) [I -41. Michaelis-Menten kinetics are obeyed at low substrate concentrations, and under such conditions the catalytic reaction proceeds by an effectively ordered mechanism with oxaloacetate adding first to the enzyme [5,6]. High concentrations of acetyl-CoA cause deviations from Michaelis-Menten kinetics, typical of substrate inhibition, in the reaction catalyzed by the pig-heart enzyme [4]. This kinetic pattern was recently shown to be due to intervention of the alternative pathway for ternary-complex formation in which acetyl-CoA binds first to the enzyme [7]. Comparison between rate constants in the two pathways to the ternary enzyme-substrate complex led to the conclusion that the affinity of citrate synthase for either substrate increases at least 20-fold on the binding of the second substrate [7], i.e. there is a strong heterotropic cooperativity in the process of substrate binding to the enzyme.

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Section: Discussionmentioning

confidence: 51%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Inhibition of Pig‐Heart Citrate Synthase by Carboxylic Acids Structurally Related to Oxaloacetate

Johansson¹,

Petterson²

1979

European Journal of Biochemistry

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The Enzymology of the Formation and Breakdown of Citrate

Srere

1975

Advances in Enzymology - And Related Areas of Molecular Biology

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Site‐Directed Spin Labelling of Nucleic Acids

Shelke

Sigurdsson

2012

Eur J Org Chem

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The study of the structure and dynamics of the nucleic acids and their complexes with other biomolecules is the basis for understanding their functions. Electron paramagnetic resonance (EPR) spectroscopy is a biophysical technique that in recent years has been increasingly used to investigate nucleic acids. EPR studies require paramagnetic centre(s), usually nitroxide spin‐label(s) that are incorporated at specific sites in the nucleic acid by site‐directed spin labelling (SDSL). In the last few years, spin labels with improved spectroscopic properties have emerged and new SDSL techniques have been developed. This microreview describes SDSL of nucleic acids in the context of the three spin labelling strategies: post‐synthetic spin labelling, labelling during oligonucleotide synthesis and noncovalent labelling.

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Interaction of a spin-labeled analog of acetyl coenzyme A with citrate synthase. Paramagnetic resonance and proton relaxation rate studies of binary and ternary complexes

Cited by 52 publications

References 35 publications

Inhibition of Pig‐Heart Citrate Synthase by Carboxylic Acids Structurally Related to Oxaloacetate

Inhibition of Pig‐Heart Citrate Synthase by Carboxylic Acids Structurally Related to Oxaloacetate

The Enzymology of the Formation and Breakdown of Citrate

Site‐Directed Spin Labelling of Nucleic Acids

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