Cooperative homotropic interaction of L-noradrenaline with the catalytic site of phenylalanine 4-monooxygenase.
Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin (S = 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20 degrees C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of L-noradrenaline to phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h = 1.9); at pH 7.2 and 20 degrees C the rat enzyme binds about 0.5 mol L-noradrenaline/mol subunit with a half-maximal binding (S50) at 0.25 microM L-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate L-phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with L-phenylalanine also leads to a complete loss of the cooperativity of L-noradrenaline binding (h = 1.0). The many similarities in binding properties of the inhibitor L-noradrenaline and the activator/substrate L-phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(III) complex.
Martínez
A
A
Department of Biochemistry, University of Bergen, Norway.
Haavik
J
J
Flatmark
T
T
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009638:Norepinephrine / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D010649:Phenylalanine / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats
D010649:Phenylalanine; D010088:Oxidoreductases; C066352:coenzyme M-7-merceptoheptanoylthreonine-phosphate-heterodisulfide hydrogenase; D010651:Phenylalanine Hydroxylase; D005033:Ethylmaleimide; D009638:Norepinephrine