tailieunhanh - Báo cáo khoa học: Protein engineering of pyruvate carboxylase Investigation on the function of acetyl-CoA and the quaternary structure

Pyruvate carboxylase (PC) fromBacillus thermodenitrificans was engineered in such away that the polypeptide chainwas 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. . | Eur. J. Biochem. 271 1391-1400 2004 FEBS 2004 doi Protein engineering of pyruvate carboxylase Investigation on the function of acetyl-CoA and the quaternary structure Shinji Sueda Md. Nurul Islam and Hiroki Kondo Department of Biochemical Engineering and Science Kyushu Institute of Technology Japan 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. Keywords acetyl-CoA biotin biotin-dependent carboxylase protein engineering pyruvate carboxylase. Pyruvate carboxylase PC is a biotin-dependent enzyme and is involved in gluconeogenesis by converting pyruvate to oxalacetate 1-3 . There are two forms of PC single polypeptide chain type and subunit type but a large majority belongs to the former class 1 4-7 . This form of PC is made of about 1200 amino acids and is distributed widely in both eukaryotes and some prokaryotes. The reaction of PC is believed to proceed in two steps just likethose of other biotin-dependent carboxylases such as acetyl-CoA carboxylase ATP HCO3 enz-biotin Đ enz-biotin-COj ADP Pi Scheme 1 enz-biotin-CO2 pyruvate Đ enz-biotin .