Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure.
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.
Sueda
Shinji
S
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Japan. sueda@bse.kyutech.ac.jp
Islam
Md Nurul
MN
Kondo
Hiroki
H
D000105:Acetyl Coenzyme A / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002262:Carboxy-Lyases / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010071:Oxaloacetates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011766:Pyruvate Carboxylase / Q000737:chemistry*; D013997:Time Factors
D010071:Oxaloacetates; D001710:Biotin; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D002262:Carboxy-Lyases; D011766:Pyruvate Carboxylase