Extrinsic Thermostabilization Factors and Thermodenaturation Mechanisms for Phosphoenolpyruvate Carboxylase (PEPC)from an Extremely Thermophilic Bacterium Phodothermus obanensis

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概要

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• The thermodenaturation of phosphoenolpyruvate carboxylase (PEPC) from an extremely thermophilic bacterum Phodothermus obamensis, capable of growth at temperature up to 85℃, was probed at different denaturation temperatures by UV-visible absorption, fluorescence emission and 1-aniliononaphthalene-8-sulfonate (ANS) binding and renaturation was assessed from diffeerent states of denaturation. Under severe denatueation conditions at 100℃, the enzyme was rapidly inactivated and its global structure immediately reached the irreversibly aggregated state by passing through the dissociated and the putative scrambled states as observed by UV-visible absorption spectroscopy. However, under milder conditions of denaturation at 93℃, the enzyme was gradually inactivated, and its global structure shifted sequentially from the dissociated state to the scrambled state. At 80℃, about 50% of the activity was left and no apparent change in the global structure occured even after 30 h. in addition, ANS binding to the enzyme was greatly increased in accordance with the change in global structure. This implies that the hydrophobic regions of the enzyme tend to be exposed to solvent due to thermal dissociation and unfolding. The extrinsic thermostabilization factors that enhance the thermpstability of the enzyme successfully suppress the thermodenaturation of the enzyme, especially the dissociation of its tetrameric form. Of these factors, the substrate for the enzyme, phosphoenolpyruvate (PEP), causes the reassociation of the dissociated inactive form of the enzyme to the active form. These results suggest that the global thermodenaturation of the enzyme results from the temperature-dependent shift of three different states and that extrinsic thermostabilization factors act to a large extent to maintain quaternary structure.

• 社団法人日本生物工学会の論文
• 1997-10-25

著者

• Yoshida Seiji

  Research Institute Of Technology Konoike Construction Co. Ltd.

• SAKO Yoshihiko

  Laboratory of Marine Microbiology, Department of Applied Bioscience, Graduate School of Agriculture,

• UCHIDA Aritsune

  Laboratory of Marine Microbiology, Department of Applied Bioscience, Graduate School of Agriculture,

• Takai Ken

  Deep-sea Microorganisms Research Group Japan Marine Science & Technology Center

• Sako Y

  Graduate School Of Agriculture Kyoto University

• Sako Yoshihiko

  Laboratory Of Microbiology Department Of Fisheries Faculty Of Agriculture Kyoto University

• Sako Yoshihiko

  Laboratory Of Marine Microbiology Division Of Applied Biosciences Graduate School Of Agriculture Kyo

• Uchida A

  Graduate School Of Agriculture Kyoto University

• Uchida Aritsune

  Laboratory Of Marine Microbiology Department Of Applied Bioscience Graduate School Of Agriculture Ky

• TAKAI KEN

  Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, K

• Sako Yoshihiko

  Laboratory Of Marine Microbiology Department Of Applied Bioscience Graduate School Of Agriculture Ky

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