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1.
The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.  相似文献   

2.
I A Rose  D J Kuo 《Biochemistry》1992,31(25):5887-5892
The amino acid decarboxylases that use an intrinsic pyruvoyl cofactor have been viewed in terms of the pyridoxal-P paradigm whereby a Schiff base is formed between the enzyme-bound cofactor and the substrate, setting up a cation sink for electrons of the C alpha-CO2- bond, ejecting CO2, and the reversal of these steps with a proton with overall retention stereochemistry. With histidine decarboxylase (pyruvoyl) it is found that the presence of CO2 is required for T-exchange between histamine and water. Since the forward reaction including formation of the C-H bond does not require added CO2, it might be assumed that the CO2 that is formed in the fragmentation step is retained by the enzyme perhaps to assist in proton transfer. No such requirement for CO2 has been reported for the pyridoxal-P-dependent decarboxylases which are generally thought to liberate CO2 prior to proton transfer. In seeking a connection between bound CO2 and proton transfer in the histidine decarboxylase reaction, one is reminded of the carboxybiotin enzymes also known for an invariant stereochemistry of retention and for the requirement that the biotin be in the carboxylated form for H-exchange to occur. Perhaps the bound CO2 of histidine decarboxylase forms a carbamate by addition to Lys155 or to an amide group of the active site. The new carboxy group could then be the vehicle for protonating the carbon from which it originated, giving overall retention of the stereochemistry at the alpha-C.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
4.
A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.  相似文献   

5.
The synthesis of several N-(5'-phosphopyridoxyl)-amino acids is described. These compounds, analogs of the Schiff base intermediate involved in enzyme-catalyzed decarboxylation, are potent inhibitors of the cognate amino acid decarboxylases. Kinetic studies using partially purified rat liver ornithine decarboxylase, have shown that N-(5'-phosphopyridoxyl)-ornithine inhibits the enzyme in a non-competitive manner with respect to both ornithine and pyridoxal-5'-phosphate. These findings suggest that the inhibitor binds to the holoenzyme active site in place of the Schiff base intermediate.  相似文献   

6.
Xiong H  Stanley BA  Pegg AE 《Biochemistry》1999,38(8):2462-2470
S-Adenosylmethionine decarboxylase is a pyruvate-dependent enzyme. The enzyme forms a Schiff base with substrate, S-adenosylmethionine, through the pyruvoyl moiety. This facilitates the release of CO2 from the substrate, which must then be protonated on the alpha carbon in order to permit hydrolysis of the Schiff base to release the product. The catalytic mechanism of human S-adenosylmethionine decarboxylase was investigated via mutagenic and kinetic approaches. The results of enzyme kinetic studies indicated that Cys-82 is a crucial residue for activity and this residue has a basic pKa. Iodoacetic acid inhibited wild-type enzyme activity in a time- and pH-dependent manner but did not affect the already reduced activity of mutant C82A. Reaction of this mutant with iodoacetic acid led to approximately one less mole of reagent being incorporated per mole of enzyme alphabeta dimer than with wild-type S-adenosylmethionine decarboxylase. Both wild-type and C82A mutant S-adenosylmethionine decarboxylases were inactivated by substrate-mediated transamination, but this reaction occurred much more frequently with C82A than with wild-type enzyme. A major proportion of the recombinant C82A mutant protein was in the transaminated form in which the pyruvoyl cofactor is converted into alanine. This suggests that incorrect protonation of the pyruvate, rather than the substrate, occurs much more readily when Cys-82 is altered. On the basis of these results, it was postulated that residue Cys-82 may be the proton donor of the decarboxylation reaction catalyzed by S-adenosylmethionine decarboxylase.  相似文献   

7.
Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.  相似文献   

8.
The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.  相似文献   

9.
We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.  相似文献   

10.
L M Abell  M H O'Leary 《Biochemistry》1988,27(16):5933-5939
The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.  相似文献   

11.
We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.  相似文献   

12.
The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis.  相似文献   

13.
Histamine is an important chemical mediator for a wide variety of physiological reactions. l-Histidine decarboxylase (HDC) is the primary enzyme responsible for histamine synthesis and produces histamine from histidine in a one-step reaction. In this study, we determined the crystal structure of human HDC (hHDC) complexed with the inhibitor histidine methyl ester. This structure shows the detailed features of the pyridoxal-5'-phosphate inhibitor adduct (external aldimine) at the active site of HDC. Moreover, a comparison of the structures of hHDC and aromatic l-amino acid (l-DOPA) decarboxylase showed that Ser-354 was a key residue for substrate specificity. The S354G mutation at the active site enlarged the size of the hHDC substrate-binding pocket and resulted in a decreased affinity for histidine, but an acquired ability to bind and act on l-DOPA as a substrate. These data provide insight into the molecular basis of substrate recognition among the group II pyridoxal-5'-phosphate-dependent decarboxylases.  相似文献   

14.
In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H2O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.  相似文献   

15.
A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.  相似文献   

16.
The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.   相似文献   

17.
The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.  相似文献   

18.
Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.  相似文献   

19.
Biosynthetic ornithine decarboxylase was purified 4300-fold from Escherichia coli to a purity of approximately 85% as judged by polyacrylamide gel electrophoresis. The enzyme showed hyperbolic kinetics with a Km of 5.6 mM for ornithine and 1.0 micronM for pyridoxal phosphate and it was competitively inhibited by putrescine and spermidine. The biosynthetic decarboxylase was compared with the biodegradative ornithine decarboxylase [Applebaum, D., et al. (1975), Biochemistry 14, 3675]. Both enzymes were dimers of 80 000-82 000 molecular weight and exhibited similar kinetic properties. However, they differed significantly in other respects. The pH optimum of the biosynthetic enzyme was 8.1, compared with 6.9 for the biodegradative. Both enzymes were activated by nucleotides, but with different specificity. Antibody to the purified biodegradative ornithine decarboxylase did not cross-react with the biosynthetic enzyme. The evolutionary relationship of these two decarboxylases to the other amino acid decarboxylases of E. coli is discussed.  相似文献   

20.
Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5'-phosphate as a cofactor, both enzymes were similar.  相似文献   

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