Inhibitors of purified beef adrenal tyrosine hydroxylase.

Two classes of compounds have been investigated as inhibitors of purified beef adrenal tyrosine hydroxylase. Among the aromatic amino acids tyrosine analogues were found to be most potent, particularly those having an a-methyl or 3-halogen substitution. Two normal metabolites, monoand diiodo-tyrosine, were found to bevery effective inhibitors. Inhibition by the amino acid analogues was shown to be competitive with the substrate. Catechols were also inhibitory, particularly 3,4-dihydroxyphenylpropyla~tamide (compound H 2254). Inhibition by the latter was reversed by cofactor (tetrahydrofolate or DMPH4) but not by tyrosine. SYMPATHETICALLY innervated tissues have been shown to contain a specific r.,-tyrosine hydroxylase which catalyzes the conversion of tyrosine to dopa,* the first step in norepinephrine biosynthesis.1 This enzyme has been purified from beef adrenal medulla and shown to require a tetrahydropte~dine~ cofactor.2 Several inhibitors of this enzyme were reported previously.2 Some were subsequently shown to inhibit tyrosine hydroxylase in vivo and to lower endogenous tissue norepinephrine levels in intact animals.3 The present report is a detailed study of two classes of tyrosine hydroxylase ~hibitors, aromatic amino acids and catechols, in which the purified beef adrenal enzyme was used. MATERIALS We wish to thank the following for making available certain compounds used in this investigation : Merck Sharp & Dohme, for the a-methyl-tyrosine, a-methyl-dopa (Aldomet), 3-iodo-a-methyl-tyrosine, 3-bromo-a-methyl-tyrosine, 3-chloro-u-methyltyrosine, and a-methyl-m-tyrosine; Ciba, Basle, for a-methyl-3-chloro-4-methoxyphenylalanine, 3-fluoro-a-methyl-tyrosine and 3-chloro-a-methyl-tyrosine. Psychopharmacology Service Center, National Institute of Mental Health, for 3,~d~ydroxyphenylacetamide; Warner-Lambert Research Institute for 3-chloro+tyrosine; Hassle Laboratories, Sweden, for 3,4-dihydroxyphenylpropylacetamide (H 22/54) and related compounds. The decarboxylase inhibitor, p-bromo-m-hydroxybenzoxyamine, was kindly supplied by Smith, Nephews, Ltd. Other chemicals were obtained from commercial sources. * Dopa: 3,4_dihydroxyphenylalanine. t The requirement of mammalian liver phenylalanine hydroxylase for reduced pteridines and the nature of these pteridines is discussed by S. Kaufman in Oxygenases, 0. Hayaishi, Chap. 4, pp. 12980, Academic Press, New York (1963). 837 838 SIDNEY UDENFRIEND, P. ZALTZMAN-NIRJWBIXG and T. NAGATSU L-Tyrosine-3,5-sH was obtained both from New England Nuclear Corp. (5,600 mc/ mmole) and Nuclear Chicago Corp. (2,000 mc/mmole). 3-Iodo-L-tyrosine-139 and diiodo-L-tyrosine-1311 were obtained from Nuclear Chicago and were prepared in the Radiochemical Center, Amersham; cc-methyl-DL-tyrosine-3H (8.1 mc/mmole) was a gift from Merck Sharp & Dohme. Each of the radioactive compounds was purified by passage over an alumina column after adjustment to pH 8.4, and the effluents were subsequently filtered through IRC-50 (Na+) columns, buffered at pH 6.5. The radioactive amino acids were, in some instances, diluted by adding nonradioactive material. Tetrahydrofolic acid was prepared according to the procedure of O’Dell et aZ.,4 and stored in vacm at -20”. 2-Amino-4-hydroxy-6,7_dimethylpteridine (DMP) was kindly supplied by Dr. L. Ellenbogen of Lederle Laboratories and reduced to the corresponding tetrahydropteridine (DMPH4) according to the procedure of Pohland et aZ.5 and stored at -20”. The latter part of the studies was carried out with DMPH4 purchased from Calbiochem. Tyrosine hydroxylase was prepared from beef adrenal medulla according to the procedure of Nagatsu et al.2 The partially purified enzyme was suspended in 0.005 M KP04 buffer, pH 7.5 ,containing 5 x 10-S M mercaptoethanol, divided into 2-ml portions and stored at -20”.