Protein engineering of pyruvate carboxylase

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.