Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC.

Area of Science:

• Biochemistry
• Enzymology
• Antioxidant Defense

Background:

• Peroxiredoxins are crucial cysteine-dependent enzymes reducing hydroperoxides.
• 2-Cys peroxiredoxins utilize a resolving cysteine to form and break disulfide bonds.
• Catalytic cycle steps require structural rearrangements for enzyme function.

Purpose of the Study:

• To determine the rate constants for individual steps in the Salmonella typhimurium AhpC catalytic cycle.
• To investigate the kinetics of peroxide reduction and disulfide bond recycling.
• To understand substrate binding effects on peroxiredoxin efficiency.

Main Methods:

• Kinetic analysis of Salmonella typhimurium AhpC.
• Fluorescence monitoring of conserved Trp residues during catalysis.
• Evaluation of rate constants for substrate binding, sulfenic acid generation, and disulfide bond formation/reduction.

Main Results:

• AhpC disulfide reduction by AhpF is a rapid, single-step process (k = 2.3 × 10(7) M(-1) s(-1)).
• Peroxide reduction involves three steps: reversible H2O2 binding (k1 = 1.36 × 10(8) M(-1) s(-1), k-1 = 53 s(-1)), sulfenic acid generation (620 s(-1)), and rate-limiting disulfide formation (75 s(-1)).
• Enzyme efficiency (kcat/Km) is primarily influenced by substrate binding rates for bulkier hydroperoxides.

Conclusions:

• Salmonella typhimurium AhpC exhibits some of the fastest catalytic rates reported for peroxiredoxins.
• The enzyme efficiently handles both the oxidative and reductive phases of its catalytic cycle.
• Substrate binding kinetics play a key role in determining the turnover efficiency of AhpC.