Abstract
Rat liver phenylalanine hydroxylase that has been activated with lysolecithin catalyzes the hydroxylation of 4-methylphenylalanine in the presence of a pterin cofactor. Two products, 4-hydroxymethylphenylalanine and 3-methyltyrosine, can be detected. The total amount of amino acids hydroxylated is equal to the amount of tetrahydropterin oxidized. Isotopic labeling studies with 18O2 and H2(18)O show that the hydroxyl groups of both products are derived from molecular oxygen and not from water. Results obtained with 2H-labeled substrates support the conclusion that these products are formed via different mechanistic pathways. Our previous investigations on substrate analogs, as well as the present results, indicate that a highly reactive oxygen-containing intermediate, such as an enzyme-bound iron-oxo compound, must be the hydroxylating species. Our present results could stimulate further discussion of the possibility that the reaction mechanism for the "NIH-shift" of the methyl group may not involve the spontaneous opening of an epoxide intermediate.
Highlights
Rat liver phenylalanine hydroxylase that has been oratory [7] showed that phenylalanine hydroxylase can activated with lysolecithin catalyzes the hydroxylation catalyze the hydroxylation of a number of other nonaromatic of 4-methylphenylalanine in the presence of a pterin amino acids such as norleucine
The last two compounds had to be reduced first by standard methods, and the tutedphenylalanines, e.g. 4-chlorophenylalanine [5] or 4- alcohols obtained were eventuallyconverted tothe corresponding methylphenylalanine [6]. The latter compoundwas found to benzyl bromideswith phosphorus tribromide1.,4-Benzenedirnethanol be hydroxylated by both bacterial (Pseudomonasspecies) and was transformed to themonobromide by a method described in [12], rat liver phenylalanine hydroxylase to two major products, 4hydroxymethylphenylalanine and 3-methyltyrosine, and one minor one, 3-hydroxy-4-methylphenylalanine(p-methyl-mtyrosine)
Essentially pure phenylal- The reactionisinhibited at higher concentrations (20.5 anine hydroxylase wasisolated from rat liver by a combination mM)ofGMPH
Summary
Our previous investigations on substrate analogs, as highest available purity. Tetrahydrobiopterin and 6-methyltetrahywell as the present results, indicate that a highly re- dropterin were purchased from B. Schirks Laboratories, Jona, Switactive oxygen-containing intermediate, such as an en- zerland. Zyme-bound iron-oxo compound, must be the hydroxylating species. Enzymes further discussion of the possibility that the reaction Catalase andsuperoxide dismutase were obtained from Boehringer mechanism for the “NIH-shift” of the methyl group Mannheim. Phenylalanine hydroxylase was prepared from rat liver may notinvolve the spontaneousopening of an epoxide by a modification of the procedure [8]of Shiman et al [9]. Teridine reductase was purified from sheep liver according to a published procedure [10]
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