Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function.

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein-cofactor interactions

Background:

• Flavin cofactors enable diverse enzymatic catalysis.
• Flavoproteins require precise control mechanisms to regulate their function.
• The protein environment dictates flavin cofactor activity and substrate specificity.

Purpose of the Study:

• To investigate the role of substrate functional groups in controlling iodotyrosine deiodinase activity.
• To elucidate the mechanism by which substrates stabilize the flavin semiquinone intermediate.
• To understand the structure-activity relationships governing reductive dehalogenation.

Main Methods:

• Enzyme kinetics assays (kcat/Km) were performed with modified halotyrosine substrates.
• Redox titrations were used to monitor flavin semiquinone accumulation.
• Binding affinities were assessed using substrate analogues lacking key functional groups.

Main Results:

• The alpha-ammonium group is essential for substrate binding and dehalogenation.
• The carboxylate group significantly impacts dehalogenation efficiency (kcat/Km > 2000-fold suppression).
• Flavin semiquinone stabilization requires coordination with both alpha-ammonium and carboxylate groups.

Conclusions:

• Substrate coordination dictates the catalytic chemistry of iodotyrosine deiodinase.
• The alpha-ammonium group is critical for enzyme-substrate complex formation and catalysis.
• Perturbations to substrate functional groups reveal alternative catalytic activities, such as nitroreductase activity.