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A general method for the analysis of random bisubstrate enzyme mechanisms

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Journal of Industrial Microbiology and Biotechnology

Abstract

In the present communication, a general method for the kinetic analysis of random bisubstrate mechanisms is described. The method comprises a stepwise application of the following kinetic and ligand-binding experiments: determination of steady-state kinetic constants, product inhibition patterns, maximum rate relationships, application of alternate substrates, application of dead-end inhibitors, direct binding of substrates, kinetic isotope effects, and isotope exchange studies. This general method was applied to a practical example: a yeast alcohol dehydrogenase-catalyzed oxidation of 2-propanol by NAD+ at pH 7.0, 25°C. It was found that this fully reversible reaction proceeds by a steady-state random Bi-Bi mechanism, whereby both dead-end complexes are formed.

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Acknowledgements

This work was financially supported by the Ministry of Science and Technology of the Republic of Serbia, Research Grant No. 1394.

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Authors and Affiliations

  1. Faculty of Technology, Bulevar Cara Lazara 1, 21000, Novi Sad, Yugoslavia

    Vladimir Leskovac & Draginja Peričin

  2. Faculty of Science, Bulevar Cara Lazara 1, 21000, Novi Sad, Yugoslavia

    Svetlana Trivić

  3. Faculty of Agriculture, Bulevar Cara Lazara 1, 21000, Novi Sad, Yugoslavia

    Julijan Kandrač

Authors
  1. Vladimir Leskovac
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  2. Svetlana Trivić
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  3. Draginja Peričin
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  4. Julijan Kandrač
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Correspondence to Vladimir Leskovac.

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Leskovac, V., Trivić, S., Peričin, D. et al. A general method for the analysis of random bisubstrate enzyme mechanisms. J IND MICROBIOL BIOTECHNOL 31, 155–160 (2004). https://doi.org/10.1007/s10295-004-0128-7

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  • DOI: https://doi.org/10.1007/s10295-004-0128-7

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