NADH Peroxidase - Reaction Mechanism

Reaction Mechanism

The NADH peroxidase from Enterococcus faecalis is unique in that it utilizes the Cys42 thiol/sulfenic acid (-SH/-SOH) redox couple in the heterolytic cleavage of the peroxide bond to catalyze the two-electron reduction of hydrogen peroxide to water.

The kinetic mechanism of the wild-type peroxidase involves (1) NADH reduction of E(FAD, Cys42-SOH) to EH2(FAD, Cys42-SH) in an initial priming step; (2) rapid binding of NADH to EH2; (3) reduction of H2O2 by the Cys42-thiolate, yielding E•NADH; and (4) rate-limiting hydride transfer from bound NADH, regenerating EH2. No discrete FADH2 intermediate has been observed, however, and the precise details of Cys42-SOH reduction have not been elucidated.

  1. E + NADH (EH2'•NAD+)* EH2'•NAD+ EH2 + NAD+ + H2O
  2. EH2 + NADH EH2•NADH*
  3. EH2•NADH* + H2O2 E•NADH + H2O
  4. E•NADH + H+ EH2•NAD+ + H2O
  5. EH2•NAD+ EH2 + NAD+

Inhibitors include Ag+, Cl-, Co2+, Cu2+, Hg2+, NaN3, Pb2+, and SO42-. At suboptimal H2O2 concentrations and concentrations of NADH that are saturating, NADH inhibits the peroxidase activity of the NADH peroxidase by converting the enzyme to an unstable intermediate. NAD+ behaves as an activator by reversing the equilibria that lead to the unstable intermediate, thus converting the enzyme to the kinetically active complex that reduces H2O2.

Read more about this topic:  NADH Peroxidase

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