A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

L-[2,5-H2]phenylalanine, an alternate substrate for rat liver phenylalanine hydroxylase

The phenylalanine analogue L-[2,5-H2]phenylalanine (1) was found to be a viable substrate (KM = 0.45 mM, kcat = 8 s-1) for L-phenylalanine-activated, rat liver phenylalanine hydroxylase (EC 1.14.16.1) (PAH). The PAH-catalyzed oxidation of 1 was stoichiometric with the oxidation of cofactor, 6-methyl-tetrahydropterin. Spectral and chromatographic data of the product from the oxidation of 1 by PAH were found to be in accord with a 3,4-epoxide. The enzymatic epoxidation of 1 is consistent with the hypothesis of an intermediate arene oxide on the reaction coordinate for PAH hydroxylation of L-phenylalanine.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Hydroxylation of 4-methylphenylalanine by rat liver phenylalanine hydroxylase

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.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Pyrimidines as cofactors for phenylalanine hydroxylase

It has been generally assumed that a tetrahydropterin (2-amino-5,6,7,8-tetrahydro-4-pteridinone) is essential for activity of the three aromatic amino acid hydroxylases. In this report it is shown that appropriately substituted pyrimidines can assume the role of cofactor for phenylalanine hydroxylase. 2,5,6-Triamino-4-pyrimidinone(V) and 5-benzylamino-2,6-diamino-4-pyrimidinone(VI) possess the same K_m values (0.1 mM and 0.003 mM) and stoichiometry of tyrosine generated to cofactor consumed (0.4 and 1.0) as their corresponding pteridine analogs, tetrahydropterin(III) and 6-phenyltetrahydropterin(IV). However, the rates with pyrimidines are lower. The ratio of rates $\text{V}\text{III}\text{= 0.045}$ and $\text{VI}\text{IV}\text{= 0.015}$. These results indicate that pteridine carbons 6 and 7 are not fundamental to cofactor binding or function, though they markedly influence the maximum velocity of hydroxylation. Pyrimidine cofactors of phenylalanine hydroxylase are valuable probes for the elucidation of the binding forces, transition states, and mechanism of oxygen activation of these hydroxylases.     

Biopterin metabolism and phenylalanine hydroxylase activity during early liver regeneration

Rat liver biopterin content and the activities of two enzymes involved in biopterin metabolism, sepiapterin reductase and dihydropteridine reductase, were not altered twenty-four hours after partial hepatectomy. This surgical procedure did, however, produce a vigorous regenerative response as verified by an increase in ornithine decarboxylase activity. The tetrahydrobiopterin-dependent activity of phenylalanine hydroxylase was increased in homogenates of regenerating liver. The pteridine requirements for the expression of this activation, and the behavior of the enzyme on calcium-phosphate cellulose columns suggest that elevated levels of cyclic adenosine monophosphate in regenerating liver induce phosphorylation and activation of phenylalanine hydroxylase. This increase in the activity of the primary enzyme of phenylalanine catabolism was interpreted as a compensatory response designed to maintain homeostasis prior to liver regeneration.     

Studies on the phenylalanine hydroxylase system in liver slices.

A method was developed to study the unsupplemented phenylalanine hydroxylase system in rat liver slices. All of the components of the system--tetrahydrobiopterin, dihydropteridine reductase, and the hydroxylase itself--are present under conditions which should be representative of the actual physiological state of the animal. The properties of the system in liver slices have been compared to those of the purified enzyme in vitro. The three pterins, tetrahydrobiopterin, 6,7-dimethyltetrahydropterin, and 6-methyltetrahydropterin, all stimulate the hydroxylation of phenylalanine when added to the liver slice medium in the presence of a chemical reducing agent. The relative velocities found at 1 mM phenylalanine and saturating pterin concentrations are: tetrahydrobiopterin, 1; 6,7-dimethyltetrahydropterin, 2.5; 6-methyltetrahydropterin, 13. This ratio of activities is similar to that found for the purified, native phenylalanine hydroxylase and indicates that the enzyme in vivo is predominantly in the native form. Rats pretreated with 6-methyltetrahydropterin showed enhanced phenylalanine hydroxylase activity in liver slices demonstrating for the first time that an exogenous tetrahydropterin can interact with the phenylalanine hydroxylase system in vivo. This finding opens up the possibility of treating phenylketonurics who still possess some residual phenylalanine hydroxylase activity with a tetrahydropterin like 6-methyltetrahydropterin which can give a large increase in rate over that seen with the natural cofactor, tetrahydrobiopterin.     

Mechanism of phenylalanine regulation of phenylalanine hydroxylase

The mechanism of phenylalanine regulation of rat liver phenylalanine hydroxylase was studied. We show that phenylalanine "activates" phenylalanine hydroxylase, converting it from an inactive to active form, by binding at a true allosteric regulatory site. One phenylalanine molecule binds per enzyme subunit; it remains at this site during catalytic turnover and, while there, cannot be hydroxylated. Loss of phenylalanine from the site causes a loss of enzymatic activity. The rate of loss of activation is dramatically slowed by phenylalanine, which kinetically "traps" activated enzyme during relaxation from the activated to unactivated state. An empirical equation is presented which allows calculation of relaxation rates over a wide range of temperatures and phenylalanine concentrations. Kinetic trapping by phenylalanine is a novel effect. It was analyzed in detail, and its magnitude implied that phenylalanine activation involves cooperativity among all four subunits of the enzyme tetramer. A regulatory model is presented, accounting for the properties of the phenylalanine activation reaction in the forward and reverse directions and at equilibrium. Fluorescence quenching studies confirmed that activation increases the solvent accessibility of the enzyme's tryptophan residues. Physical and kinetic properties of purified phenylalanine hydroxylase from rat, rabbit, baboon, and goose liver were compared. All enzymes were remarkably alike in catalytic and regulatory properties, suggesting that control of this enzyme is similar in mammals and birds.