Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

Stereoselective effects in the interactions of pterin cofactors with rat-liver phenylalanine 4-monooxygenase

The (6R) and (6S) epimers of l-erythro-tetrahydrobiopterin (BH4) and some of its structural analogs, were tested as cofactors and non-covalent effectors in the phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) reaction. The oxidation-reduction potentials (Em,7) of the free (not enzyme-bound) form of the (6R) and (6S) epimers were rather similar (range 174-184 mV) for the oxidation of tetrahydropterins to quinonoid dihydropterins. Rapid-mixing kinetic experiments were performed at 20 degrees C under conditions which allow only a few turnover reactions of the enzyme. Three main oxidation products were identified spectroscopically at pH 6.8 for all three tetrahydropterins tested: the C(4a)-hydroxy derivatives, the quinonoid dihydropterins, and the stable 7,8-dihydropterins (in that sequence). The formation of the C(4a)-hydroxy forms closely paralleled that of tyrosine, and supports the proposal that this covalent adduct is formed as an immediate product on completion of the catalytic cycle. Assay of the initial rate of C(4a)-hydroxy derivative formation represents a new approach in kinetic studies of this enzyme, and the kinetic parameters obtained for the phenylalanine-activated enzyme are presented. The affinity of binding of (6R)-BH4 and (6S)-BH4 to phenylalanine hydroxylase was also estimated on the basis of their quenching of the intrinsic tryptophan fluorescence of the enzyme. The apparent affinities were found to correspond well to the Km values estimated in kinetic studies of the hydroxylation reaction with the phenylalanine activated enzyme, i.e. higher for (6R)-BH4 than for (6S)-BH4. The lower V value observed for the native enzyme with the (6R) epimer in steady-state kinetics is explained by its higher potency as a negative effector, since the oxidation-reduction potentials of the two diastereomers were similar. Dihydrobiopterin (BH2) was found to inhibit the hydroxylation reaction and quenched the intrinsic tryptophan fluorescence of the enzyme with the same concentration dependence as that observed with (6S)-BH4.     

The effects of tetrahydroisoquinolinecarboxylic acids on tyrosine 3-monooxygenase

The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor (K_i = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor (K_i = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid (K_i = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid (K_i = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro, decreases in the K_i of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the K_i of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC₅₀ = 100 μm) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo.     

A comparative study of tyrosine 3-monooxygenase from rat adrenal and brainstem

Spleen cells from a CBF1 (BALB/c X C57BL/6) mouse immunized with rat tyrosine 3-monooxygenase were fused with NS-1 mouse myeloma cells. From 188 hybrid cells, 2 stable clones secreting anti-tyrosine 3-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful for isolating rat tyrosine 3-monooxygenase from crude preparations. Analyses by monoclonal antibody chromatography followed by SDS-polyacrylamide gel electrophoresis and by gel filtration revealed that tyrosine 3-monooxygenases from nerve cell bodies, nerve terminals, and adrenal medullae were indistinguishable with respect to their molecular structures. However, there were serious differences in the catalytic properties between the enzymes from the brain tissues and adrenal medullae, although there appeared to be no significant difference between the enzymes from nerve cell bodies and nerve terminals. The possibility that the activity of the enzyme may be strongly suppressed especially at the physiological pH in brain tissues is also discussed.     

Evidence for involvement of protein kinase in the activation by adenosine 3':5'-monophosphate of brain tyrosine 3-monooxygenase.

Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase.     

The mechanism of alpha-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase. What is the role of quinonoid intermediates?

To shed light on the mechanism of isotopic exchange of alpha-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their alpha-deuterated analogues in D2O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the alpha-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of alpha-proton exchange.     

Aminoacrylate intermediates in the reaction of Citrobacter freundii tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the alpha-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an alpha-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted alpha-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-l-cysteines, l-tyrosine, and 3-fluoro-l-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the alpha-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at approximately 365 nm. The value of the rate constant for aminoacrylate formation is similar to k(cat), suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-l-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the alpha-aminoacrylate is observed, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibit kinetic isotope effects of approximately 2-3 on alpha-aminoacrylate formation when the alpha-(2)H-labeled substrates are used, consistent with the previously reported internal return of the alpha-proton to the phenol product. These results are the first direct spectroscopic observation of alpha-aminoacrylate intermediates in the reactions of TPL.     

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.     

Reduced 6,6,8-trimethylpterins. Preparation, properties and enzymic reactivities with dihydropteridine reductase, phenylalanine hydroxylase and tyrosine hydroxylase

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8-dihydro(6 H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8-trimethyl-5,6,7,8-tetrahydro(3 H)pterin was synthesised and the quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin derived from it is extremely stable with a half-life in 0.1 M Tris/HCl (pH 7.6, 25 degrees C) of 33 h. Quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin is not a substrate for dihydropteridine reductase but it is reduced non-enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 microM, pH 7.6, 25 degrees C when using quinonoid 6-methyl-7,8-dihydro(6 H)pterin as substrate. 6,6,8-Trimethyl-5,6,7,8-tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.     

Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation.

1. Melanosomal tyrosinase was isolated from normal C57B1 mice, and a comparison of the tyrosine-hydroxylation and dopa (3,4-dihydroxyphenylalanine)-oxidation activities of this enzyme was made. 2. The results indicate that in the absence of dopa cofactor, this enzyme is capable of tyrosine hydroxylation, but with very little subsequent dopa oxidation and melanin formation. 3. This mechanism of enzyme action may play an important role in the intracellular regulation of melanin formation. 4. Further, dopa appears to act as a positive allosteric effector for tyrosine hydroxylation by tyrosinase, in addition to its known activity as a hydrogen donor for the reaction.     

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.     

Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

The cross-linking of tyrosine by treatment with tetranitromethane

1. Tyrosine was treated with tetranitromethane. 2. Approx. 10% of the tyrosine was converted into 3-nitrotyrosine. 3. Three fluorescent compounds were also formed. They appear to be a dimer, trimer and tetramer in which tyrosine units are linked by biphenyl bonds. 4. The dimer and trimer have also been isolated from some proteins after treatment with tetranitromethane. 5. The yield of 3-nitrotyrosine from ovotransferrin after treatment with tetranitromethane was much smaller than the loss of tyrosine. 6. Several unidentified compounds were also formed by the reaction between tyrosine and tetranitromethane.     

Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate

Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase. According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor. The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine. The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL. This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method. For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction. Both effects are due to destabilization of the quinonoid and they counterbalance each other. Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change. In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate.     

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.     

Oxygen dependence of tyrosine hydroxylase

Summary. The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, ³H₂O from 3,5-³H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1–2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K _m-values of 29–45 μM for dioxygen were determined for all human TH isoforms, i.e. 2–40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the K _m (O₂)-values had declined to an average of 20 ± 4 μM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities. Authors’ address: Jan Haavik, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway     

Catalytic residues of the telomere resolvase ResT: a pattern similar to, but distinct from, tyrosine recombinases and type IB topoisomerases

ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of co-valently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5'-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4-6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.     

A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization

Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.