Seymour Kaufman

Phenylketonuria: Biochemical Mechanisms

As implied by the title, this review of phenylketonuria (PKU) will be neither comprehensive nor encyclopedic. Rather, it will be limited to those aspects of the disease where sufficient biochemical knowledge is available to support meaningful discussion—admittedly, often speculative—about underlying mechanisms. Recent comprehensive reviews are available (Hsia, 1970; Knox, 1972).

Evidence for the lack of direct phosphorylation of bovine caudate tyrosine hydroxylase following activation by exposure to enzymatic phosphorylating conditions.

Abstract Highly purified bovine caudate tyrosine hydroxylase can be activated by exposure to enzymatic phosphorylating conditions. This activation is due to both a decrease in the K m for the pterin cofactor and to some increase in V max . The K m for the enzymes substrate, tyrosine, is unchanged by activation. After tyrosine hydroxylase was activated in the presence of [γ- 32 P]-ATP, no incorporation of 32 P into the enzyme was observed by either immunoprecipitation studies or by sucrose gradient studies.

Interaction of Phosphorylated Tryptophan Hydroxylase with 14-3-3 Proteins

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25–30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by ∼45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (K a ) of 4.5 × 107 m −1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation.

Parallel Induction of Nitric Oxide and Tetrahydrobiopterin Synthesis by Cytokines in Rat Glial Cells

Abstract: Activation of monocyte‐derived macrophages with cytokines leads to the induction of nitric oxide synthase. Much less is known about the effects of cytokines on microglia, resident brain macrophages, or on astrocytes. In this study, we compared the induction by lipopolysaccharide, interferon‐γ, and tumor necrosis factor‐α of nitric oxide production and synthesis of tetrahydrobiopterin, the required cofactor for nitric oxide synthase, in microglia and peritoneal macrophages. Activation of microglia induced parallel increases in nitric oxide and intracellular tetrahydrobiopterin levels, although induction of the latter appears to be somewhat more sensitive to diverse stimulators. As with macrophages, inducible nitric oxide production in microglia was blocked by inhibitors of tetrahydrobiopterin biosynthesis. Interleukin‐2, an important component of the neuroimmunomodulatory system, was only a weak activator of microglia by itself but potently synergized with interferon‐γ to stimulate production of both nitric oxide and tetrahydrobiopterin. Astrocytes were also activated by lipopolysaccharide and combinations of cytokines but showed a somewhat different pattern of responses than microglia. Biopterin synthesis was increased to higher levels in astrocytes than in microglia, but maximal induction of nitric oxide production required higher concentrations of cytokines than microglia and the response was much lower. These results suggest that tetrahydrobiopterin synthesis in glial cells is a potential target for therapeutic intervention in acute CNS infections whose pathology may be mediated by overproduction of nitric oxide.

Studies on phenylalanine and tyrosine hydroxylation by rat brain tyrosine hydroxylase.

Tyrosine hydroxylase (EC1.14.16.2), presumably the rate-limiting enzyme in the biosynthesis of catecholamines, is known to catalyze the hydroxylation of both phenylalanine and tyrosine. Using both an isolated enzyme preparation and a synaptosomal preparation, where some architectural integrity of the tissue has been preserved, we have attempted to evaluate the manner in which these two substrates are hydroxylated by rat brain tyrosine hydroxylase. In the presence of tetrahydrobiopterin the isolated enzyme catalyzes the hydroxylation of phenylalanine to 3,4-dihydroxyphenylalanine with the release of free tyrosine as an obligatory intermediate. In contrast, the rat brain striatal synaptosomal preparation in the presence of endogenous cofactor converts phenylalanine to 3,4-dihydroxyphenylalanine without the release of free tyrosine.

Activation of tyrosine hydroxylase by polyanions and salts. An electrostatic effect.

The activity of a partially purified preparation of tyrosine hydroxylase (EC 1.14.16.2) from the bovine caudate nucleus was increased by heparin, chondroitin sulfate, phosphatidylserine, polyacrylic acid, polyvinyl sulfuric acid and both poly-D-, and poly-L-glutamic acids, all polyanions. A variety of salts both activated the enzyme and prevented the activation by the polyanions. The observations that activity is increased when the enzyme interacts with salts and with macromolecules of high negative charge density are used to infer a model for these interactions and for the structural change in the enzyme that accompanies activation.

Phenylalanine hydroxylase activity in liver biopsies from hyperphenylalaninemia heterozygotes: deviation from proportionality with gene dosage.

Extract: Liver biopsy samples from the patients with hyperphenylalaninemia have an average of 5% of the normal hydroxylase activity. The parents of the patients have between 7.3% (excluding the value for one parent) and 10% of the normal hepatic hydroxylase activity. An explanation for these findings involves negative interallelic complementation, which involves protein-protein interaction between subunits in a multitneric enzyme. In support of this model is the evidence that rat liver phenylalanine hydroxylase is a multimeric protein composed of two electrophoretically distinguishable subunits.Speculation: The finding that parents of patients with hyperphenylalaninemia have an average of 10% of the normal level of hepatic phenylalanine hydroxylase, a multimeric enzyme, can be explained on the assumption that the liver tissue of heterozygotes has an excess of enzyme molecules that contain at least one mutant subunit.

Phenylalanine hydroxylase of human liver: Assay and some properties

Abstract A sensitive, reliable assay for phenylalanine hydroxylase in human liver extracts is described. A few milligrams of tissue are sufficient for a quantitative determination of the enzyme activity in normal liver. With this procedure, the apparent Km values for phenylalanine and for 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, a cofactor analogue, have been determined for the human liver enzyme.

Effect of Cyclic AMP‐Dependent Protein Phosphorylating Conditions on the pH‐Dependent Activity of Tyrosine Hydroxylase from Beef and Rat Striata

Abstract Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23‐fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP‐dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G‐25 Sephadex‐treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP‐dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.

Determination of the phosphate content of purified proteins

A technique is described which allows accurate and reproducible determinations of the amount of phosphate that is covalently bound to purified protein. The procedure involves the removal of free or noncovalently bound phosphate, an accurate estimation of the protein concentration of the washed sample, ashing of the sample, and subsequent phosphate measurement. The technique enables the reliable quantitation of subnanomole levels of phosphate.