Magnetic resonance studies on the copper site of dopamine beta-monooxygenase in the presence of cyanide and azide anions.
In order to elucidate the coordination state of water molecules in the Cu(II) site of dopamine [( 3,4-dihydroxyphenyl)ethylamine] beta-monooxygenase, measurements of the paramagnetic 1H nuclear magnetic relaxation rate of solvent water in the enzyme solution containing cyanide or azide as an exogenous ligand were carried out to obtain the values of intrinsic paramagnetic relaxation rate decrements Rp1 and Rp2 for the ligand-enzyme 1:1 and 2:1 complexes, respectively. Rp1 (percent) values were 53 (pH 5.5) and 52 (pH 7.0) for cyanide and 38 (pH 5.5) and 32 (pH 7.0) for azide, while Rp2 (percent) values were 98 (pH 5.5) and 96 (pH 7.0) for azide. Although no Rp2 values for cyanide were obtained because of its reducing power at the Cu(II) site, the Rp1 and Rp2 values obtained above prove that the Cu(II) center has two coordinated water molecules that are exchangeable for exogenous ligands at either pH. Supporting evidence was provided by electron paramagnetic resonance (EPR) titration, in which the enzyme solution containing cyanide-enzyme (1:1) complex in an equal proportion to uncomplexed enzyme gave an observed paramagnetic relaxation rate decrement, Rp, of 23%. Another characteristic of the Rp1 and Rp2 values was their invariability with respect to pH, indicating that the three-dimensional structure of the Cu(II) site is pH-invariant within the range examined. Binding constants of ligand to enzyme Kb1 and Kb2 for 1:1 and 2:1 complex formation, respectively, were also determined through an analysis of the Rp values; it was found that Kb1 was larger than Kb2 irrespective of pH. (ABSTRACT TRUNCATED AT 250 WORDS)
Obata
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Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan.
Tanaka
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Kawazura
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D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D003300:Copper / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008962:Models, Theoretical; D019810:Sodium Azide; D012966:Sodium Cyanide / Q000494:pharmacology*
D001386:Azides; D003486:Cyanides; D003300:Copper; D019810:Sodium Azide; D004299:Dopamine beta-Hydroxylase; D012966:Sodium Cyanide