The effect of ligands of phenylalanine 4-monooxygenase on the cAMP-dependent phosphorylation of the enzyme.

Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection

We have found that rat liver phenylalanine hydroxylase oxidizes a stoichiometric amount of its cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH4), in a reaction that is independent of phenylalanine. The reaction requires oxygen, and one 6MPH4 is oxidized per subunit of enzyme. A quinonoid dihydropterin is directly produced in the reaction, and there is no evidence for the intermediate formation of a 4a-hydroxydihydropterin. Neither hydrogen peroxide nor superoxide anions were detected as products of the oxidation, and phenylalanine hydroxylase appears to be the sole electron acceptor from 6MPH4. Therefore, in a functional sense, phenylalanine hydroxylase is reduced by its cofactor. The reduced state of the enzyme is stable to activation by phenylalanine and during catalytic turnover, and the electrons on the reduced enzyme cannot be directly used to drive phenylalanine hydroxylation. Of greatest importance, enzyme reduction appears to be required for the formation of a catalytically active enzyme species. Phenylalanine hydroxylase is chemically and physically altered by reduction. Reduced enzyme exhibits (1) a greatly increased fluorescence, which is quantitatively related to the extent of reduction, (2) an altered UV-visible absorbance spectrum, (3) a greatly increased sensitivity to inactivation by hydrogen peroxide, and (4) a greatly decreased sensitivity to inhibition by Dopa which quantitatively correlates with the increase in enzyme fluorescence. Second-order rate constants, kr, for the reduction of the enzyme by 6MPH4 have been determined and found to vary with pH, temperature, buffer, and enzyme activation: at pH 6.8, 25 degrees C, and in phosphate buffer, for phenylalanine-activated enzyme kr = 15 X 10(6) min-1 M-1. Tris is a competitive inhibitor with respect to 6MPH4 of enzyme reduction and also of catalysis.

Stoichiometric reduction of phenylalanine hydroxylase by its cofactor: a requirement for enzymatic activity.

The tautomeric structure of the cofactor product of aromatic amino acid hydroxylases, quinoid dihydrobiopterin, is still unknown. Characterization of this molecule, which is also the substrate for dihydropteridine reductase (EC 1.6.99.7), has been hindered by the rapid rearrangement of quinoid dihydropterins to 7,8-dihydropterins. This tautomerization can be prevented by disubstitution at the 6-position. A procedure is presented for the synthesis of 6,6-disubstituted pterins from a vicinal diamine and 2-amino-6-chloro-4(3H)-pyrimidinone. The method is illustrated with the specific synthesis of 6,6-dimethyltetrahydropterin (6,6-Me2PH4). 6,6-Me2PH4 is a cofactor for rat liver phenylalanine hydroxylase (EC 1.14.16.1), with enzyme kinetic parameters similar to those of its positional isomer, 6,7-dimethyltetrahydropterin. The resulting quinoid 6,6-dimethyldihydropterin (q-6,6-Me2PH2) is stable; the half-life in 0.1 M Tris-HCl, pH 7.4, at 27 and 37 degrees C is 4 and 1.25 h, respectively. q-6,6-Me2PH2, produced either by phenylalanine hydroxylase or by chemical oxidation of 6,6-Me2PH4, is a substrate for dihydropteridine reductase, with a Km of 0.4 mM and a maximum velocity double that of the natural isomer of quinoid dihydrobiopterin. In concentrations up to 0.4 mM q-6,6-Me2PH2 is not an inhibitor of phenylalanine hydroxylase, in contrast to 6-methyl-7,8-dihydropterin and 7,8-dihydrobiopterin which inhibit competitively, with Ki's of 0.2 mM and 0.05 mM, respectively. The stability of q-6,6-Me2PH2 has facilitated definitive determination of chemical and physical properties of a quinoid dihydropterin.

6,6-Dimethylpterins: stable quinoid dihydropterin substrate for dihydropteridine reductase and tetrahydropterin cofactor for phenylalanine hydroxylase

The in vivo rate of conversion of phenylalanine to tyrosine (PheOH) can be estimated using combinations of stable isotope-labeled phenylalanine and tyrosine. We have compared in four healthy adult men the rates of phenylalanine conversion to tyrosine based on the following pairs of primed, continuous tracer infusions administered simultaneously: (1) L-[ring-2H5]phenylalanine and 2H2-tyrosine with a 2H4-tyrosine prime, and (2) L-[1-13C]phenylalanine and 2H2-tyrosine with a 1-13C-tyrosine prime. Phenylalanine oxidation was determined from measurement of 13CO2 excretion in expired air. Tracers were given for 8 hours, with subjects being in the postabsorptive state during the first 3 hours and in the fed state during the remaining 5 hours. Mean (+/- SD) rates (mumol.kg-1.h-1) of phenylalanine conversion to tyrosine for fasted and fed states, respectively, were 5.1 +/- 2.9 and 6.8 +/- 3.4 with 2H5-phenylalanine and significantly higher (P .1) from the rate of phenylalanine conversion to tyrosine determined using 13C-phenylalanine. These results indicate the need for caution in interpreting kinetic aspects of phenylalanine metabolism when based on isotopic data from multideuterated phenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylalanine conversion to tyrosine: Comparative determination with l-[ring-2H5]phenylalanine and l-[1-13C]phenylalanine as tracers in man

An assay for picomole levels of tyrosine and related phenols and its application to the measurement of phenylalanine hydroxylase activity

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An assay for picomole levels of tyrosine and related phenols and its application to the measurement of phenylalanine hydroxylase activity