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Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

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Abstract

Pyruvate kinase acts as an allosteric enzyme, playing a crucial role in the catalysis of the final step of the glycolytic pathway. In this study, site-specific mutagenesis and tryptophan fluorescence quenching were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Movement of the B domain was found to be essential for the catalytic reaction. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft. Trp-157 accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481 and Trp-514 are brought into an aqueous environment after phenylalanine binding.

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Abbreviations

PK:

Pyruvate kinase

CD:

Circular dichroism

FT-IR:

Fourier transform infrared spectroscopy

ADP:

Adenosine diphosphate

PEP:

Phosphoenolpyruvate

TMK buffer:

50 mM Tris, 8 mM MgSO4, and 75 mM KCl at pH 7.5

Phe:

Phenylalanine

GdnHCl:

Guanidine hydrochloride

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Acknowledgments

This project was supported in part by grants from the National Natural Science Foundation of China (No. 30970631), and Shanghai Leading Academic Discipline Project (No. B109).

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Authors and Affiliations

  1. Department of Chemistry, Fudan University, Shanghai, 200433, China

    Feng Li, Ting Yu, Yuwei Zhao & Shaoning Yu

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  1. Feng Li
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  2. Ting Yu
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  3. Yuwei Zhao
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  4. Shaoning Yu
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Corresponding author

Correspondence to Shaoning Yu.

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Feng Li and Ting Yu equally contribute to this work.

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Li, F., Yu, T., Zhao, Y. et al. Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching. Eur Biophys J 41, 607–614 (2012). https://doi.org/10.1007/s00249-012-0828-2

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  • DOI: https://doi.org/10.1007/s00249-012-0828-2

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