Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase

Authors

Joshua H. Hakala, Marquette University
Amanda J. Laseke, Marquette University
Anya Lei Koza, Marquette University
Martin St. Maurice, Marquette UniversityFollow

Document Type

Article

Publication Date

8-2022

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

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.

Comments

Accepted version. Biochemistry, Vol. 61, No. 17 (2022): 1824-1835. DOI. © 2022 American Chemistry Society. Used with permission.

Recommended Citation

Hakala, Joshua H.; Laseke, Amanda J.; Koza, Anya Lei; and St. Maurice, Martin, "Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase" (2022). Biological Sciences Faculty Research and Publications. 928.
https://epublications.marquette.edu/bio_fac/928