Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure. |
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Authors: | Shinji Sueda Md Nurul Islam Hiroki Kondo |
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Affiliation: | Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Japan. sueda@bse.kyutech.ac.jp |
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Abstract: | Pyruvate carboxylase (PC) from Bacillus thermodenitrificans was engineered in such a way that the polypeptide chain was divided into two, between the biotin carboxylase (BC) and carboxyl transferase (CT) domains. The two proteins thus formed, PC-(BC) and PC-(CT+BCCP), retained their catalytic activity as assayed by biotin-dependent ATPase and oxamate-dependent oxalacetate decarboxylation, for the former and the latter, respectively. Neither activity was dependent on acetyl-CoA, in sharp contrast to the complete reaction of intact PC. When assessed by gel filtration chromatography, PC-(BC) was found to exist either in dimers or monomers, depending on the protein concentration, while PC-(CT + BCCP) occurred in dimers for the most part. The two proteins do not associate spontaneously or in the presence of acetyl-CoA. Based on these observations, this paper discusses how the tetrameric structure of PC is built up and how acetyl-CoA modulates the protein structure. |
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