A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.     

Homology between phenylalanine and tyrosine hydroxylases reveals common structural and functional domains

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TYH) are mixed-function oxidases that share many characteristic biochemical and immunological properties. The recent cloning and sequencing of full-length cDNAs for both human PAH and rat TYH allow detailed comparison of their primary structures. There is a high degree of homology between PAH and TYH on nucleic acid and amino acid levels. The pattern of homology suggests that these molecules are comprised of a homologous core containing the determinants for enzymatic activity and a nonhomologous region that contributes to substrate specificity and regulation. The degree of homology also suggests that these two proteins evolved from a common ancestor.     

Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.     

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.     

Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.     

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.     

Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase

Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for L-tyrosine versus L-phenylalanine as a substrate. The pH-dependence of TAL activity with L-tyrosine and L-phenylalanine demonstrates a common protonation state for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid substrates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of MIO-dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of RsTAL and related MIO-containing enzymes.     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent. This complicated catalytic process has diverse requirements that are difficult to be satisfied by a single site. Two general strategies have evolved to satisfy these requirements. para-Hydroxybenzoate hydroxylase, the paradigm for the single-component flavoprotein monooxygenases, is one of the most thoroughly studied of all enzymes. This enzyme undergoes significant protein and flavin dynamics during catalysis. There is an open conformation that gives access of substrate and product to solvent, and a closed or in conformation for the reaction with oxygen and the hydroxylation to occur. This closed form prevents solvent from destabilizing the hydroperoxyflavin intermediate. Finally, there is an out conformation achieved by movement of the isoalloxazine toward the solvent, which exposes its N5 for hydride delivery from NAD(P)H. The protein coordinates these dynamic events during catalysis. The second strategy uses a reductase to catalyze the reduction of the flavin and an oxygenase that uses the reduced flavin as a substrate to react with oxygen and hydroxylate the organic substrate. These two-component systems must be able to transfer reduced flavin from the reductase to the oxygenase and stabilize a C4a-peroxyflavin until a substrate binds to be hydroxylated, all before flavin oxidation and release of H2O2. Again, protein dynamics are important for catalytic success.     

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.     

Distinct forms of the protein kinase-dependent activator of tyrosine and tryptophan hydroxylases.

Tyrosine and tryptophan hydroxylases are the key enzymes in the regulation of catecholamine and serotonin levels in neurons and other endocrine cells. Among the mechanisms proposed for the modulation of activity, phosphorylation of the enzyme is believed to be of functional significance with respect to the stimulus-response coupling, but the precise mechanism is unknown. Here, we show the existence of multiple, distinct forms of the 14-3-3 activator protein, a neuronal protein essential for activation of tyrosine and tryptophan hydroxylases by Ca2+/calmodulin-dependent protein kinase type II. Bovine brain 14-3-3 protein was resolved by reversed-phase chromatography into seven polypeptides (alpha to eta), all of which were active towards tryptophan hydroxylase when the renatured preparations were assayed in the presence of Ca2+, calmodulin and the protein kinase. Determination of the amino acid sequences of the beta and gamma chains and comparison of the sequences with the previously determined sequence of the eta chain revealed that these molecules are highly homologous, and share a common structural feature in containing an extremely acidic C-terminal region predicted as a domain for interaction with the phosphorylated hydroxylases. Northern blot analysis indicated that the beta, gamma and eta chain are expressed abundantly in the brain; however, these polypeptides appear to be expressed with different tissue specificities because gamma mRNA is found only in the brain, while lower levels of beta and eta mRNAs are detected in several other tissues. These findings suggest the involvement of a diverse family of the activator protein in the stimulus-coupled, Ca2(+)-dependent regulation of monoamine biosynthesis.