Abstract
The acetate kinase from the Antarctic psychrophilic Shewanella sp. AS-11 (SAK) has a significantly higher catalytic efficiency at low temperatures when compared with that from mesophilic Escherichia coli K-12 (EAK). To examine the stability and conformational flexibility of SAK and EAK, steady state intrinsic fluorescence studies were performed. EAK contains only one Trp at a position 46, while SAK contains two Trps at positions 46 and 388. From the fluorescence emission spectra, quenching with acrylamide, Cs+ and I− at different temperatures and denaturation with guanidine-HCl, it was revealed that the SAK bears more flexible and unstable structure than that of EAK. Substrate-induced conformational changes reflect that SAK reached transition state through more conformational changes than EAK. In combination of our thermodynamic studies on the enzymatic reaction and present research findings, it can be concluded that these structural features of SAK may contribute to its high catalytic efficiency at low temperatures.
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Abbreviations
- ASKHA:
-
Acetate and sugarkinases/Hsc70/actin
- EAK:
-
Acetate kinase from Escherichia coli K-12
- SAK:
-
Acetate kinase from Shewanella species AS-11
- HEPES:
-
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
- GdmCl:
-
Guanidine-HCl
- Trp:
-
Tryptophan
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Acknowledgments
We would like to thank Dr. Masao Tokunaga and Dr. Yukio Nagano for their interest, encouragement, and valuable discussion. This study was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science to KW (15380074) and by Rendai-student Supporting Program of the United Graduate School of Agricultural Sciences, Kagoshima University to AKT.
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Tang, M.A.K., Motoshima, H. & Watanabe, K. Fluorescence Studies on the Stability, Flexibility and Substrate-Induced Conformational Changes of Acetate Kinases from Psychrophilic and Mesophilic Bacteria. Protein J 31, 337–344 (2012). https://doi.org/10.1007/s10930-012-9408-7
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DOI: https://doi.org/10.1007/s10930-012-9408-7