Pyruvate carboxylase (PC) is a biotin‐dependent, homotetrameric enzyme that is responsible for the MgATP‐dependent catalytic conversion of pyruvate to oxaloacetate in the presence of the essential allosteric activator, acetyl‐CoA. PC is a critical anaplerotic enzyme in glucose metabolism, serving to replenish oxaloacetate consumed by the TCA cycle. Increased production of acetyl‐CoA, due to high lipid supply, has been shown to upregulate PC activity in the liver, contributing to the development of obesity‐induced insulin resistance and Type 2 Diabetes. Previous crystallographic studies have identified four distinct domains that comprise the PC monomer, namely the biotin carboxylase (BC), biotin carboxyl carrier (BCCP), carboxyl transferase (CT), and allosteric domains. Each domain has been functionally characterized via steady‐state kinetics and site‐directed mutagenic studies of catalytically relevant residues. The tetramer is a dimer of dimers, whereby each face is composed of a dimer of monomers arranged antiparallel to each other.Pyruvate binding has been shown to trigger the movement of the BCCP domain to an opposing polypeptide chain, though it is not known whether this is facilitated through an inter‐or intramolecular “signal.” That is, does pyruvate binding on the same monomer or on the opposing polypeptide chain facilitate the intermolecular BCCP domain translocation? To determine whether BCCP domain movements are governed by intermolecular or intramolecular mechanisms, we have generated PC hybrid tetramers (HTs) consisting of two strategically chosen mutants: one with an inactive BC domain and wild‐type CT domain (E218A); and the other with a wild‐type BC domain and a mutant CT domain (T882S) exhibiting a 62‐fold decrease in kcat and 3‐fold increase in Km, pyruvate as compared to wild‐type. Steady‐state kinetics of the full‐forward reaction catalyzed by the T882S:E218A(1:1) HT demonstrate that the pyruvate concentration needed to facilitate BCCP translocation is significantly higher than that required for the carboxylation reaction. More importantly, our results indicate that the occupancy of the CT domain by pyruvate on an opposing polypeptide chain “triggers” the translocation of the BCCP domain from the BC domain on a monomer to the opposing CT domain. Further study of additional molecular interactions within the tetrameric structure, such as BC—BC domain interactions between faces of the enzyme, will be used to elucidate the nature of the mechanism by which PC controls BCCP translocation and the coordination of catalysis between two spatially distinct active sites.
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