Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase

Biochemistry. 2022 Sep 6;61(17):1824-1835. doi: 10.1021/acs.biochem.2c00298. Epub 2022 Aug 9.


Biotin-dependent enzymes employ a carrier domain to efficiently transport reaction intermediates between distant active sites. The translocation of this carrier domain is critical to the interpretation of kinetic and structural studies, but there have been few direct attempts to investigate the dynamic interplay between ligand binding and carrier domain positioning in biotin-dependent enzymes. Pyruvate carboxylase (PC) catalyzes the MgATP-dependent carboxylation of pyruvate where the biotinylated carrier domain must translocate ∼70 Å from the biotin carboxylase domain to the carboxyltransferase domain. Many prior studies have assumed that carrier domain movement is governed by ligand-induced conformational changes, but the mechanism underlying this movement has not been confirmed. Here, we have developed a system to directly observe PC carrier domain positioning in both the presence and absence of ligands, independent of catalytic turnover. We have incorporated a cross-linking trap that reports on the interdomain conformation of the carrier domain when it is positioned in proximity to a neighboring carboxyltransferase domain. Cross-linking was monitored by gel electrophoresis, inactivation kinetics, and intrinsic tryptophan fluorescence. We demonstrate that the carrier domain positioning equilibrium is sensitive to substrate analogues and the allosteric activator acetyl-CoA. Notably, saturating concentrations of biotin carboxylase ligands do not prevent carrier domain trapping proximal to the neighboring carboxyltransferase domain, demonstrating that carrier domain positioning is governed by conformational selection. This model of carrier domain translocation in PC can be applied to other multi-domain enzymes that employ large-scale domain motions to transfer intermediates during catalysis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetyl-CoA Carboxylase / metabolism
  • Biotin / chemistry
  • Carboxyl and Carbamoyl Transferases* / genetics
  • Carboxyl and Carbamoyl Transferases* / metabolism
  • Catalytic Domain
  • Ligands
  • Pyruvate Carboxylase* / chemistry
  • Staphylococcus aureus


  • Ligands
  • Biotin
  • Carboxyl and Carbamoyl Transferases
  • Pyruvate Carboxylase
  • Acetyl-CoA Carboxylase