Design and kinetic characterization of multisubstrate inhibitors of dopamine .beta.-hydroxylase

• Published in: Biochemistry (Easton) Vol. 25; no. 23; pp. 7271 - 7278
• Main Authors: Kruse, Lawrence I, DeWolf, Walter E, Chambers, Pamela A, Goodhart, Paula J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18.11.1986
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Chromaffin Granules - enzymology; dopamine beta -monooxygenase; Dopamine beta-Hydroxylase - antagonists & inhibitors; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Imidazoles - chemical synthesis; Imidazoles - pharmacology; Indicators and Reagents; Kidney Medulla - enzymology; Kinetics; Mathematics; Oxidoreductases; peptide synthesis; Structure-Activity Relationship; Dopamine β-monooxygenase; Vertebrata; Mammalia; Enzyme; Enzyme inhibitor; Ox; Renal medulla; Artiodactyla; Kinetics; Ungulata
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00371a004

The synthesis and kinetics characterization of a new class of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) inhibitor, 1-(4-hydroxybenzyl)imidazole-2-thiol, is reported. These inhibitors, which incorporate a phenethylamine substrate mimic and an oxygen mimic into a single molecule, exhibit both the kinetic properties and the potency (Kis approximately 10(-9) M) expected for a multisubstrate inhibitor and are therefore classified as such. Steady-state kinetic experiments with these multisubstrate inhibitors and their substructural analogues support the recently proposed pH-dependent changes in substrate binding order [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3106] and a mechanism whereby the inhibitor binds specifically to the reduced Cu+ form of enzyme at both the phenethylamine substrate site and the active-site copper atom(s). A Yonetani-Theorell double-inhibition experiments indicates mutually exclusive binding of the inhibitor substructures p-cresol and 1-methylimidazole-2-thiol to suggest an extremely short intersite distance between the phenethylamine binding site and the active-site copper atom(s).