Closed fumarase C active-site structures reveal SS Loop residue contribution in catalysis

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Fumarase C (FumC) is crucial for the citric acid cycle, catalyzing fumarate to S-malate conversion.
• The enzyme superfamily exhibits a dynamic SS Loop, whose active-site placement is critical for catalytic function.
• Active-site asymmetry in FumC raises questions about SS Loop's influence on residue participation.

Purpose of the Study:

• To elucidate the structural and catalytic roles of SS Loop residues (S318, K324, N326) in Escherichia coli FumC.
• To investigate the impact of SS Loop variations on FumC's active-site conformations and reaction kinetics.

Main Methods:

• High-resolution X-ray crystallography was employed to determine FumC active-site structures.
• Kinetic analyses were performed on FumC variants to assess enzyme activity and substrate interaction.

Main Results:

• Three distinct FumC active-site conformations were identified: disordered-open, ordered-open, and a novel ordered-closed state.
• Mutations in SS Loop residues did not alter Michaelis constants (Km) but significantly reduced turnover numbers (kcat).

Conclusions:

• The SS Loop plays a significant role in modulating FumC's catalytic efficiency rather than substrate binding affinity.
• Specific residues within the SS Loop (S318, K324, N326) are proposed to have defined structural and catalytic functions in FumC.
• The newly discovered ordered-closed conformation offers new insights into FumC's reaction mechanism.