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Kinetic Properties of Two Rhizopus Exo-polygalacturonase Enzymes Hydrolyzing Galacturonic Acid Oligomers Using Isothermal Titration Calorimetry

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Abstract

The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a K m of 55 μM and k cat of 10.3 s−1 while RPG16 was shown to have greater affinity for (GalpA)2 with a K m of 16 μM, but lesser catalytic activity with a k cat of 3.9 s−1. Both enzymes were inhibited by the product, galacturonic acid, with app K i values of 886 and 501 μM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)3 with a K m of 9.2 μM and a similar k cat at 10.7 s−1 relative to (GalpA)2. Catalytic constants for RPG16 hydrolyzing (GalpA)3 could not be determined; however, single-injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)3 relative to (GalpA)2. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes.

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References

  1. Niture, S. K. (2008). Biologia, 63, 1–19.

    Article  CAS  Google Scholar 

  2. Markovič, O., & Janaček, Š. (2001). Protein Engineering, 14, 615–631.

    Article  Google Scholar 

  3. Henrissat, B., & Davies, G. (1997). Current Opinion in Structural Biology, 7, 637–644.

    Article  CAS  Google Scholar 

  4. Zhang, J., Henriksson, H., Szabo, I. J., Henriksson, G., & Johansson, G. (2005). Journal of Industrial Microbiology and Biotechnology, 32, 431–438.

    Article  CAS  Google Scholar 

  5. Stotz, H. U., Bishop, J. G., Bergmann, C. W., Koch, M., Albersheim, P., Darvill, A. G., et al. (2000). Physiological and Molecular Plant Pathology, 56, 117–130.

    Article  CAS  Google Scholar 

  6. Kars, I., Krooshof, G. H., Wagemakers, L., Joosten, R., Benen, J. A. E., & van Kan, J. A. L. (2005). The Plant Journal, 43, 213–225.

    Article  CAS  Google Scholar 

  7. Kester, H. C. M., & Visser, J. (1990). Biotechnology and Applied Biochemistry, 12, 150–160.

    CAS  Google Scholar 

  8. Kluskens, L. D., van Alebeek, G. W. M., Walther, J., Voragen, A. G. H., de Vos, W. M., & van der Oost, J. (2005). FEBS Journal, 272, 5464–5473.

    Article  CAS  Google Scholar 

  9. Kester, H. C. M., Kusters-Van Someren, M. A., Müller, Y., & Visser, J. (1996). European Journal of Biochemistry, 240, 738–746.

    Article  CAS  Google Scholar 

  10. Miller, G. L. (1959). Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  11. Somogyi, M. (1952). Journal of Biological Chemistry, 153, 375–380.

    Google Scholar 

  12. Breuil, C., & Saddler, J. N. (1985). Enzyme and Microbial Technology, 7, 327–332.

    Article  CAS  Google Scholar 

  13. Baumann, M. J., Murphy, L., Lei, N., Krogh, K., Borch, K., & Westh, P. (2011). Analytical Biochemistry, 410, 19–26.

    Article  CAS  Google Scholar 

  14. Mertens, J. A., & Bowman, M. J. (2011). Current Microbiology, 62, 1173–1178.

    Article  CAS  Google Scholar 

  15. Jordan, D. B., Mertens, J. A., & Braker, J. D. (2009). Biochimica et Biophysica Acta, 1794, 144–158.

    Article  CAS  Google Scholar 

  16. Krokeide, I., Synstad, B., Gåseidnes, S., Horn, S. J., Eijsink, V. G. H., & Sørlie, M. (2007). Analytical Biochemistry, 363, 128–134.

    Article  CAS  Google Scholar 

  17. Dennhart, N., Fukamizo, T., Brzezinski, R., Lacombe-Harvey, M., & Letzel, T. (2008). Journal of Biotechnology, 134, 253–260.

    Article  CAS  Google Scholar 

  18. Todd, M. J., & Gomez, J. (2001). Analytical Biochemistry, 296, 179–187.

    Article  CAS  Google Scholar 

  19. Krokeide, I., Eijsink, V. G. H., & Sørlie, M. (2007). Thermochim. Acta, 454, 144–146.

    Article  CAS  Google Scholar 

  20. Karim, N., & Kidokoro, S. (2004). Thermochim. Acta, 412, 91–96.

    Article  CAS  Google Scholar 

  21. Jeoh, T., Baker, J. O., Ali, M. K., Himmel, M. E., & Adney, W. S. (2005). Analytical Biochemistry, 347, 244–253.

    Article  CAS  Google Scholar 

  22. Bohlin, C., Olsen, S. N., Morant, M. D., Patkar, S., Borch, K., & Westh, P. (2010). Biotechnology and Bioengineering, 107, 943–952.

    Article  CAS  Google Scholar 

  23. Mertens, J. A., Hector, R. E., & Bowman, M. J. (2012). Thermochim. Acta, 527, 219–222.

    Article  CAS  Google Scholar 

  24. Gill, S. C., & von Hippel, P. H. (1989). Analytical Biochemistry, 182, 319–326.

    Article  CAS  Google Scholar 

  25. Leatherbarrow, R. J. (2001). Grafit version 5. Horley: Erithacus Software Ltd.

    Google Scholar 

  26. Cho, S. W., Lee, S., & Shin, W. (2001). Journal of Molecular Biology, 314, 863–878.

    Article  Google Scholar 

  27. van Santen, Y., Nenen, J. A. E., Schröter, K., Kalk, K. H., Armand, S., Visser, J., et al. (1999). Journal of Biological Chemistry, 274, 30474–30480.

    Article  Google Scholar 

  28. Norberg, A. L., Karlsen, V., Hoell, I. A., Bakke, I., Eijsink, V. G. H., & Sørlie, M. (2010). FEBS Letters, 585, 4581–4585.

    Article  Google Scholar 

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  1. Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, 1815 North University Street, Peoria, IL, 61604, USA

    Jeffrey A. Mertens

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  1. Jeffrey A. Mertens
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Correspondence to Jeffrey A. Mertens.

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Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

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Mertens, J.A. Kinetic Properties of Two Rhizopus Exo-polygalacturonase Enzymes Hydrolyzing Galacturonic Acid Oligomers Using Isothermal Titration Calorimetry. Appl Biochem Biotechnol 170, 2009–2020 (2013). https://doi.org/10.1007/s12010-013-0336-6

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