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Biochemistry. 1995 Oct 31;34(43):14114-24.

Analysis of the kinetic mechanism of enterococcal NADH peroxidase reveals catalytic roles for NADH complexes with both oxidized and two-electron-reduced enzyme forms.

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Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.


Anaerobic titrations of the two-electron-reduced NADH peroxidase (EH2) with NADH and 3-acetylpyridine adenine dinucleotide (AcPyADH) yield the respective complexes without significant formation of the four-electron-reduced enzyme (EH4). Further analysis of the EH2/EH4 redox couple, however, yields a midpoint potential of -312 mV for the free enzyme at pH 7. The catalytic mechanism of the peroxidase has been evaluated with a combination of kinetic and spectroscopic approaches, including initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzyme (E) and EH2 with NADH and AcPyADH, and diode-array spectral analyses of both the reduction of E-->EH2 by NADH and the formation of EH2.NADH. Overall, these results are consistent with rapid formation of an E.NADH complex with distinct spectral properties and a rate-limiting hydride transfer step that yields EH2, with no direct evidence for intermediate FADH2 formation. The EH2.NADH complex described previously [Poole, L. B., & Claiborne, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slowly with H2O2. Stopped-flow analyses do, however, support the very rapid formation of an EH2.NADH* intermediate, with spectral properties that distinguish it from the static EH2.NADH form, and yield a first-order rate constant for the conversion between the two species that is smaller than kcat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mechanism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R.P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167].

[Indexed for MEDLINE]

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