Km Values For Tyrosine Research Articles

Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.

Mutation of Serine 395 of Tyrosine Hydroxylase Decouples Oxygen−Oxygen Bond Cleavage and Tyrosine Hydroxylation

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase.

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.

Isolation and partial characterization of a broad specificity aminotransferase from Leishmania mexicana promastigotes

A broad specificity aminotransferase (BSAT), with high activity with both, aromatic amino acids and aspartate as substrates, was purified to homogeneity from promastigotes of Leishmania mexicana by a method involving chromatography on DEAE-cellulose, Red-120-Sepharose and Mono Q, and gel filtration on Sephacryl S-200. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, was 90 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequence of four internal peptides obtained by tryptic digestion. Two of these peptides, consisting of 49 amino acid residues in total, showed high similarity (57%) with corresponding sequences of plant aspartate aminotransferases, whereas they had only 33% identity with the aromatic aminotransferase of Escherichia coli, and 16% identity with the tyrosine aminotransferase from the related parasite Trypanosoma cruzi. The BSAT contained only one 1/2 Cys residue per monomer. The optimal pH for the enzyme reaction, with tyrosine and α-oxoglutarate as substrates, was 7.0. The apparent Km values for tyrosine, phenylalanine, tryptophan and glutamate, with oxaloacetate as co-substrate, were 1.3, 0.9, 0.9 and 171.8 mM, respectively; the value for aspartate with α-oxoglutarate as co-substrate was 2.5 mM, and that for alanine with α-oxoglutarate as co-substrate was 216 mM. The values for pyruvate, α-oxoglutarate and oxaloacetate, with tyrosine as co-substrate, were 5.6, 0.71 and 0.12 mM, respectively. These results suggest that the enzyme is a broad-specificity aminotransferase, able to transaminate the aromatic amino acids, aspartate, and to a lower extent alanine, with high sequence similarity to aspartate aminotransferases.

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.

Enhancement of In Vivo Tyrosine Hydroxylation in the Rat Adrenal Gland Under Hypoxic Conditions

We examined the effects of hypoxia (8% O2) on in vivo tyrosine hydroxylation, a rate-limiting step for catecholamine synthesis, in the rat adrenal gland. The hydroxylation rate was determined by measuring the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition. One hour after hypoxic exposure, DOPA accumulation decreased to 60% of control values, but within 2 h it doubled. At 2 h, the apparent Km values for tyrosine and for biopterin cofactor of tyrosine hydroxylase (TH) in the soluble fraction were unchanged, whereas the Vmax value increased by 30%. The content of total or reduced biopterin was unchanged, but the content of tyrosine increased by 80%. Tyrosine administration had little effect on DOPA accumulation under room air conditions but enhanced DOPA accumulation under hypoxia. After denervation of the adrenal gland, the hypoxia-induced increase in DOPA accumulation and in the Vmax value was abolished, whereas the hypoxia-induced increase in tyrosine content was persistent. These results suggest that in vivo tyrosine hydroxylation is enhanced under hypoxia, although availability of oxygen is reduced. The enhancement is the result of both an increase in tyrosine content coupled with increased sensitivity of TH to changes in tyrosine tissue content and of an increase in dependence of TH on tyrosine levels. The increase in the sensitivity of TH and in the Vmax value is neurally induced, whereas the increase in tyrosine content is regulated by a different mechanism.

Development of tyrosine aminotransferase and para-hydroxyphenylpyruvate dioxygenase activities in fetal and neonatal human liver.

In livers of fetuses of 220--340 g body wt, total cytosolic tyrosine aminotransferase activity was 1.0 nmol of product/mg of protein per min, and the corresponding values for autopsy livers of newborns of 740--1,475 g and 2,600--3,650 g were 1.5 and 5.7, respectively, as compared with the adult value of 12.7. On the other hand, para-hydroxyphenylpyruvate dioxygenase activity is at adult level already in fetuses less than 340 g body wt. The Km value for tyrosine of tyrosine aminotransferase (1 mM) was considerably higher than the corresponding value for para-hydroxyphenylpyruvate of para-hydroxyphenylpyruvate dioxygenase (50 micro M). These results suggest that tyrosine aminotransferase is the rate limiting enzyme in the catabolism of tyrosine in premature infants.

Heterogeneity of Hepatic Tyrosine Aminotransferase. Separation of the Multiple Forms from Rat and Frog Liver by Isoelectric Focussing and Hydroxylapatite Column Chromatography and Their Partial Characterization.

L-Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) and other enzymes that transaminate tyrosine in rat liver cytosol have been separated into four fractions by isoelectric focussing. One of the forms is probably identical to mitochondrial L-aspartate:2-oxoglutarate aminotransferase (EC 2.6.1.1.; mASAT). The other three forms have pI's of 4.72, 4.98 and 5.30 and Km values of 1.3 and 0.3 mM for tyrosine and alpha-ketoglutarate. These heat stable forms have little or no ASAT activity. Rat liver TAT is also separated into three peaks by hydroxylapatite. Each fraction gives only one peak of activity when electrofocussed separately. In the frog, three groups of peaks of TAT activity have been separated by hydroxylapatite column chromatography. The first group is connected with ASAT activity. These peaks (pI's 6.35, 6.50 and 6.90) are heat stable and have a Km value for tyrosine of 4 mM. These fractions probably represent cytoplasmic ASAT (sASAT). The second group of peaks has at least two subforms (pI's 9.0 and 9.4, Km for tyrosine 15 mM). These forms probably represent mASAT. The third group consists of three forms that resemble the major forms of rat liver TAT. These results indicate that heterogeneity is common to many aminotransferases and independent of regulation by glucocorticoids.

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Abstract—Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle‐bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris‐acetate buffer, and at 6·5 in Tris‐HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. Km values toward various tetrahydropteridines such as l‐erythro‐tetrahydrobiopterin (a probable natural cofactor), 2‐amino‐4‐hydroxy‐6‐methyltetrahydropteridine, and 2‐amino‐4‐hydroxy‐6,7‐dimethyltetrahydropteridine were 2 × 10−5m, 5 × 10−5m and 4 × 10−4m, respectively. The Km value for tyrosine at 1 × 10−3m‐2‐amino‐4‐hydroxy‐6‐methyltetrahydropteridine as a cofactor was 5 × 10−5m. The enzyme activity was markedly stimulated with Fe2+ or catalase, but Fe2+ gave higher activity. The activity was inhibited with α, α′‐dipyridyl, l‐α‐methyl‐p‐tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l‐5‐Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d‐5‐hydroxytryptophan nor 5‐hydroxytryptamine inhibited the enzyme. The inhibition by l‐5‐hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10−5m, and partially uncompetitive at concentrations lower than 9 × 10−5m. The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.

Purification and characterization of tyrosyl-RNA synthetase from baker's yeast.

Tyrosyl-tRNA synthetase [EC 6. 1. 1. 1] was purified from baker's yeast. The purified preparation had no cross reaction with 19 other amino acids. Three kinds of assay involving hydroxamate formation, ATP-PP1 exchange and tyrosyl-tRNA formation were adopted to characterize the enzyme. It was observed that the Km values for tyrosine obtained with the above three methods were not the same. Among many tyrosine analogues only 3-iodo-tyrosine and D-tyrosine stimulated the ATP-PP1 exchange reaction, while tyrosol and tyramine were competitive inhibitors. Based on these results it was concluded that the hydroxyl group at the para-position and the α-amino group of the substrate have an essential role in binding to the enzyme. The effects of several metal ions upon enzymatic activity were studied. The effects differed remarkably depending upon the assay methods.