Zinc Switch in Pig Heart Lipoamide Dehydrogenase: Steady-State and Transient Kinetic Studies of the Diaphorase Reaction.
Biochemistry (Mosc)
; 85(8): 908-919, 2020 Aug.
Article
en En
| MEDLINE
| ID: mdl-33045951
ABSTRACT
Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M-1·s-1, (9 ± 2) × 104 M-1·s-1, and (1.6 ± 0.5) × 105 M-1·s-1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Zinc
/
Electrones
/
Dihidrolipoamida Deshidrogenasa
/
Miocardio
/
NADH Deshidrogenasa
Límite:
Animals
Idioma:
En
Revista:
Biochemistry (Mosc)
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos