Structural studies of dopamine. beta. -hydroxylase

Thesis/Dissertation · Mon Dec 31 23:00:00 EST 1984

OSTI ID:7189160

Papadopoulos, N J

Dopamine ..beta..-hydroxylase catalyzes the conversion of dopamine to norepinephrine, a ..beta..-hydroxylation reaction, utilizing ascorbic acid as reducing agent and molecular oxygen as cosubstrate. Modifications of the previously published purification procedure for D..beta..H have produced findings which show that (1) enzyme is inactivated by ascorbate autooxidation during the isolation procedure, (2) active as well as inactive D..beta..H co-purify throughout the entire purification procedure and (3) beef liver catalase totally protects against this time dependent inactivation. The stoichiometry of copper binding to the active sites of D..beta..H has been investigated using /sup 19/F-NMR and radioactive binding experiments. The data unequivocally show that homogeneous D..beta..H (isolated in the presence of catalase) specifically binds up to approx.8 copper atoms per enzyme tetramer. Distance determinations done using NMR relaxation rate theory show that anion activators of the catalytic reaction are bound at a fairly far distance from the Cu/sup 2 +/ centers. Spin-echo electron paramagnetic resonance spectroscopy indicates that at least one, possibly two, histidines are bound as equatorial ligands to each Cu/sup 2 +/ ion. The combined data indicate that highly purified dopamine ..beta..-hydroxylase contains a 2 copper atom active site, composed of magnetically non-interacting metal centers. Active site components are distant from the Cu/sup 2 +/ centers, suggesting a possible movement of active site residues or components after reduction of enzyme bound copper in order to achieve the insertion of 1 atom of oxygen into the benzylic C-H bond of dopamine.

• Research Organization: Pennsylvania State Univ., University Park (USA)
• OSTI ID: 7189160
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL HORMONES
AMINES
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
CARDIOTONICS
CARDIOVASCULAR AGENTS
COPPER
DOPAMINE
DRUGS
ELEMENTS
ENZYMES
FLUORINE 19
FLUORINE ISOTOPES
HORMONES
HYDROXY COMPOUNDS
HYDROXYLASES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
METALS
MOLECULAR STRUCTURE
NEUROREGULATORS
NORADRENALINE
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PHENOLS
POLYPHENOLS
RESONANCE
SPIN ECHO
STABLE ISOTOPES
STEROID HORMONES
STOICHIOMETRY
SYMPATHOMIMETICS
TRANSITION ELEMENTS