Divergent Mechanisms of Allosteric Regulation of Pyruvate Carboxylase by Acetyl Coenzyme A

Pyruvate carboxylase is a biotin‐dependent enzyme, composed of multiple functional domains on a single polypeptide chain, including the biotin carboxylase domain, the carboxyltransferase domain, the biotin carboxyl carrier protein domain and the allosteric domain. Acetyl coenzyme A is an allosteric activator of pyruvate carboxylase from a wide variety of species. However, the sensitivity to acetyl coenzyme A activation varies depending on the organism from which the enzyme was cloned or isolated. We are investigating the divergent allosteric activation of pyruvate carboxylase from two organisms, Rhizobium etli ( Re PC) and Aspergillus nidulans ( An PC). The maximum activity of Re PC is accelerated ~10‐fold by acetyl coenzyme A, while An PC is insensitive to acetyl coenzyme A activation, despite binding acetyl coenzyme A. Previous studies have structurally and kinetically characterized Re PC in the presence and absence of acetyl coenzyme A. In order to investigate the divergent mechanisms of allosteric activation in An PC and Re PC, we have structurally and kinetically characterized the contributions of acetyl coenzyme A to catalytic turnover in An PC. We have observed a highly divergent response of An PC and Re PC to acetyl coenzyme A activation in both the overall pyruvate carboxylation reaction and in the half‐reactions catalyzed at the individual catalytic domains. The binding of acetyl coenzyme A to Re PC at the allosteric domain is primarily mediated by two conserved arginine residues. We generated mutations at the equivalent arginine residues in An PC and determined that the K a for acetyl coenzyme A was significantly increased in both mutations, indicating that acetyl coenzyme A most likely binds at the same binding site in An PC as it does in Re PC. To further clarify the mechanism of acetyl coenzyme A activation in pyruvate carboxylase, we investigated the catalytic turnover of hybrid tetramers composed of several combinations of inactivating mutations. Our data reveals that, in Re PC, acetyl coenzyme A constrains the translocation of the biotin carboxyl carrier protein domain, limiting it to sample only two productive motions during catalytic turnover. However, in An PC, acetyl coenzyme A does not alter the range of motions sampled by the biotin carboxyl carrier protein domain during catalysis. Thus, while acetyl coenzyme A binds at the same binding site in both An PC and Re PC, the divergent response of An PC and Re PC to acetyl coenzyme A activation arises, in part, from different innate carrier domain movements during catalytic turnover. Support or Funding Information This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01