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Sugar Binding Effects on the Enzymatic Reaction and Conformation Near the Active Site of Pokeweed Antiviral Protein Revealed by Fluorescence Spectroscopy

Journal of Fluorescence volume 24, pages 951–958 (2014)Cite this article

Abstract

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

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Abbreviations

PAP:

Pokeweed anti-viral protein

RIP:

Ribosome-inactivating protein

FRET:

Fluorescence resonance energy transfer

NAG:

N-acetylglucosamine

TCSPC:

Time-correlated single photon counting

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Affiliations

  1. Institute of Biophysics, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan

    Hiromichi Nakashima, Ryosuke Ueno & Etsuko Nishimoto

  2. Institute of Material Chemistry and Engineering, Kyushu University, Kasuga, Fukuoka, 816-8580, Japan

    Yukihiro Fukunaga

Authors
  1. Hiromichi Nakashima
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  2. Yukihiro Fukunaga
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  3. Ryosuke Ueno
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  4. Etsuko Nishimoto
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Correspondence to Etsuko Nishimoto.

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Nakashima, H., Fukunaga, Y., Ueno, R. et al. Sugar Binding Effects on the Enzymatic Reaction and Conformation Near the Active Site of Pokeweed Antiviral Protein Revealed by Fluorescence Spectroscopy. J Fluoresc 24, 951–958 (2014). https://doi.org/10.1007/s10895-014-1378-z

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  • DOI: https://doi.org/10.1007/s10895-014-1378-z

Keywords

  • Ribosome-inactivating protein
  • Pokeweed anti-viral protein
  • N-glycosidase
  • FRET
  • Fluorescence depolarization