Characterization of alternate reductant binding and electron transfer in the dopamine. beta. -monooxygenase reaction
The steady-state limiting kinetic parameters V/sub max/, V/K/sub DA/, and V/K/sub O/sub 2//, together with deuterium isotope effects on these parameters, have been determined for the dopamine ..beta..-monooxygenase (D..beta..M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascrobate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of V/sub max/ and V/K/sub DA/, and the authors conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/K/sub O/sub 2// have been measured with (2,2-/sup 2/H/sub 2/)dopamine as substrate both in the presence and in the absence of added ascorbate. This key result unambiguously rules out an entry of electrons to enzyme forms leading from the enzyme-dopamine complex to enzyme-bound product and, hence, reaction mechanisms involving a reductive activation of the putative Cu(II)-OOH prior to substrate hydroxylation.
- Research Organization:
- Univ. of California, Berkeley
- OSTI ID:
- 5965071
- Journal Information:
- Biochemistry; (United States), Vol. 26:17
- Country of Publication:
- United States
- Language:
- English
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DEUTERIUM COMPOUNDS
ISOTOPE EFFECTS
OXYGENASES
BIOCHEMICAL REACTION KINETICS
ABSORPTION SPECTROSCOPY
CATECHOLAMINES
COPPER SULFATES
DOPAMINE
ELECTRON TRANSFER
AMINES
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
CARDIOTONICS
CARDIOVASCULAR AGENTS
COPPER COMPOUNDS
DRUGS
ENZYMES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
KINETICS
NEUROREGULATORS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PHENOLS
POLYPHENOLS
REACTION KINETICS
SPECTROSCOPY
SULFATES
SULFUR COMPOUNDS
SYMPATHOMIMETICS
TRANSITION ELEMENT COMPOUNDS
550201* - Biochemistry- Tracer Techniques