Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase.

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Biotin-dependent enzymes utilize a carrier domain for efficient intermediate transfer between active sites.
• Understanding the dynamic interplay between ligand binding and carrier domain positioning is crucial for kinetic and structural studies.

Purpose of the Study:

• To directly observe carrier domain positioning in pyruvate carboxylase (PC) independent of catalytic turnover.
• To investigate the mechanism governing carrier domain translocation in PC, specifically the role of ligand binding.

Main Methods:

• Development of a cross-linking trap to monitor carrier domain proximity to the carboxyltransferase domain.
• Analysis of cross-linking by gel electrophoresis, inactivation kinetics, and intrinsic tryptophan fluorescence.

Main Results:

• Carrier domain positioning is sensitive to substrate analogues and the allosteric activator acetyl-CoA.
• Saturating biotin carboxylase ligands do not prevent carrier domain trapping, indicating conformational selection governs positioning.
• The carrier domain translocation mechanism in PC is independent of catalytic turnover.

Conclusions:

• Carrier domain positioning in pyruvate carboxylase is primarily driven by conformational selection rather than solely ligand-induced changes.
• This conformational selection model for domain translocation is applicable to other multi-domain enzymes.
• The study provides a novel method to investigate enzyme domain dynamics.