Construction of new forms of pyruvate carboxylase to assess the allosteric regulation by acetyl-CoA

Abstract

The single polypeptide chain of Bacillus thermodenitrificans pyruvate carboxylase (PC) is composed of the biotin carboxylase (BC), carboxyl transferase (CT) and biotin carboxyl carrier protein (BCCP) domains from the amino terminus. This polypeptide chain was divided into two between the CT and BCCP domains. The resulting proteins, PC-(BC + CT) and PC-(BCCP), were expressed in Escherichia coli separately, purified to homogeneity and characterized. PC-(BC + CT) was 4% as active as native PC in the carboxylation of pyruvate with PC-(BCCP) as substrate with a K(m) of 39 microM. Moreover, acetyl-CoA stimulated the carboxylation of PC-(BCCP) about 3-fold, whereas it was without effect in the corresponding reaction with free biotin. In addition to these engineered proteins, another form of enzyme was also constructed in which the BC domain of B.thermodenitrificans PC was replaced with the BC subunit of Aquifex aeolicus PC, whose activity is independent of acetyl-CoA. The resulting chimera was about 7% as active as native PC, but its activity was independent of acetyl-CoA. On the basis of these observations, the mechanism by which acetyl-CoA regulates the reaction of PC is discussed.

The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase

“…A similar biotin-dependent rate enhancement has been reported for Bacillus thermodenitrificans PC [18]. The rate enhancement is dependent on the degree to which the biotin side-chain mimics the biotinylated enzyme.…”

“…A biotin-independent oxaloacetate decarboxylation activity was first described for chicken liver PC following incubation with avidin [49]. This finding was corroborated by the report of a concentration dependent biotin-independent rate of oxaloacetate decarboxylation for chicken liver PC incubated with avidin [14] and has also been observed in B. thermodenitrificans PC in the absence of a BCCP domain[18]. However, mutations at the biotinylation site in both B. thermodenitrificans PC and Re PC completely eliminated the oxamate-induced oxaloacetate decarboxylation activity, leading to the contradictory claim that biotin is required for oxaloacetate decarboxylation in PC [12, 19].…”

“…In an earlier study, BCCP-biotin was also reported to be more effective than free biotin in enhancing the rate of oxaloacetate decarboxylation in PC from Bacillus thermodenitrificans [18]. Similarly, in E. coli carboxyltransferase, a 2000-fold increase over biotin was observed when the biotinylated C-terminal BCCP domain was used as a substrate in place of free biotin [41].…”

“…The description of oxaloacetate decarboxylation in PC is also plagued by contradictory literature reports concerning whether the biotin cofactor is essential for the decarboxylation reaction [11, 14, 16, 18, 19]. This is a crucial mechanistic question as it has significant implications on the stringency of the active site geometry and on the mode of carboxyl transfer.…”

Interaction between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli

“…Recently, Tong and coworkers reported the structure of E. coli BC complexed with free biotin and bicarbonate (39). While this structure is significant, it is unable to explain why free biotin is a relatively poor substrate compared to BCCP-biotin in both E. coli BC and PC (41, 49, 60). The anchor point from which tethered biotin accesses the BC domain active site is clearly defined in the current T882A Re PC structure.…”