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T V Demidkina

Publications and source records attributed to T V Demidkina.

At least 19 recordsLinked to original sources
PubMed2006

Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C--H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site.

In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied. The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition of l-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C-alpha-proton of a number of amino acids for deuterium in (2)H(2)O. The k(ex) values for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the k(cat) values for reacting substrates. Thus, for the mutant TPLs the stage of C-alpha-proton abstraction may be considered as a rate-limiting for the whole reaction.

Alanine↗
PubMed2006

L-methionine gamma-lyase from Citrobacter freundii: cloning of the gene and kinetic parameters of the enzyme.

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.

Amino Acid Sequence↗
PubMed2005

Structure of Citrobacter freundii L-methionine gamma-lyase.

L-Methionine gamma-lyase (MGL) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes gamma-elimination of L-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.

Bacterial Proteins↗
PubMed2003

Role of arginine 226 in the mechanism of tryptophan indole-lyase from Proteus vulgaris.

In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3;-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 10(5)-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 10(3)-10(4)-fold. The mutant enzyme catalyzed exchange of the C-alpha-proton in complexes with some inhibitors with rates reduced 10(2)-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme-inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme-substrate intermediates.

Amino Acid Sequence↗
PubMed2002

Tryptophan indole-lyase from Proteus vulgaris: kinetic and spectral properties.

An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites.

Binding Sites↗
PubMed2000

Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids.

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

Amino Acids↗
PubMed2000

Pyridoxal 5'-phoshate schiff base in Citrobacter freundii tyrosinephenol-lyase. Ionic and tautomeric equilibria.

Spectral properties of the internal Schiff base in tyrosine phenol-lyase have been investigated in the presence of an activating cation K+ and a cation-inhibitor Na+. The holoenzyme absorption spectra in the pH range 6.5-8.7 were recorded in the presence of K+. No apparent pKa value of the coenzyme chromophore was found in this pH range, indicating that the internal Schiff base does not change its ionic form on going from pH 6.5 to 8.7. To determine the ionic state and tautomeric composition of the Schiff base in tyrosine phenol-lyase, the absorption and circular dichroism spectra were analyzed using lognormal distribution curves. The predominant form of the internal Schiff base is that with protonated pyridinium and aldimine nitrogen atoms and deprotonated 3'-hydroxy group, i.e. the ketoenamine. This form is in prototropic equilibrium with its enolimine tautomer. The internal aldimine ionic form is changed upon replacement of K+ with Na+. This replacement leads to a significant decrease in the pKa value of pyridinium nitrogen of the pyridoxal-P.

Bacterial Proteins↗
PubMed2000

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.

Alanine↗
PubMed1997

The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Arginine↗
PubMed1996

Purification and crystals of tyrosine phenol-lyase from Erwinia herbicola.

New method of purification of tyrosine phenol-lyase from Erwinia herbicola has been developed. The enzyme obtained is homogeneous and characterised by a specific activity which is three times higher then that described earlier. Crystals of holoenzyme complexed with monovalent cations have been grown from NaCl, KCl and (NH4)2SO4 containing solutions. The crystals belong to P6(2)22 space group. They are stable to the X-ray radiation and diffract up to 2.6-3.1 A. Asymmetric unit contains one subunit of tetrameric molecule.

Crystallization↗
PubMed1995

Site-directed mutagenesis of tyrosine-71 to phenylalanine in Citrobacter freundii tyrosine phenol-lyase: evidence for dual roles of tyrosine-71 as a general acid catalyst in the reaction mechanism and in cofactor binding.

Tyr71 is an invariant residue in all known sequences of tyrosine phenol-lyase (TPL). The substitution of Tyr71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for beta-elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these substrates exhibit kcat values reduced by 10(3)-10(4)-fold, while the kcat/Km values are reduced by 10(2)-10(3)-fold, compared to wild-type TPL. However, for substrates with good leaving groups (S-(o-nitrophenyl)-L-cysteine,beta-chloro-L-alanine, and O-benzoyl-L-serine), Y71F TPL exhibits kcat values 1.85-7% those of wild-type TPL. Y71F TPL forms very stable quinonoid complexes with strong absorbance at 502 nm from L-phenylalanine, tyrosines (L-tyrosine, 3-fluoro-L-tyrosine, and [alpha-2H]-3-fluoro-L-tyrosine), and S-alkylcysteines (S-methyl-L-cysteine, S-ethyl-L-cysteine, and S-benzyl-L-cysteine). The time courses of the formation of quinonoid intermediates in these reactions are biphasic. The slow phase shows a dependence on concentration of PLP and is due to the cofactor binding steps, while the fast phase is due to the amino acid alpha-deprotonation and reprotonation steps. The rate constants for the fast phase of the reactions of Y71F TPL with L-phenylalanine and S-methylcysteine are similar to those for alpha-deprotonation or reprotonation steps in the reactions of wild-type TPL. The PLP binding constant of Y71F TPL is estimated to be 1 mM by spectrophotometric titration, compared to 0.6 microM for wild-type TPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Three-dimensional structure of tyrosine phenol-lyase.

Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacter freundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH4-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5'-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5'-phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-A resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the crystallographic 2-fold symmetry axis of the crystal which belongs to the space group P2(1)2(1)2 with a = 76.0 A, b = 138.3 A, and c = 93.5 A. Each subunit comprises 14 alpha-helices and 16 beta-strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5'-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5'-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.

Amino Acid Sequence↗
PubMed1992

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme.

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.

Chemical Phenomena↗
PubMed1989

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations.

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.

Cations, Monovalent↗
PubMed1988

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, beta-phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L-amino acids, D-phenylalanine and D-tryptophan are competitive inhibitors. The enzymatic isotope exchange of the alpha-proton in 2H2O was observed only in the case of L-amino acids. For L-phenylalanine and L-tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol-lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L-tyrosine, 3-fluoro-L-tyrosine), the 'key' phenol-elimination step is not the rate-limiting one, the reaction velocity being determined by the preceding alpha-proton abstraction.

Amino Acids↗
PubMed1988

Crystallization and crystal data on tyrosine phenol-lyase.

Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained.

Ammonium Sulfate↗
PubMed1987

Transamination catalysed by tyrosine phenol-lyase from Citrobacter intermedius.

The interactions of tyrosine phenol-lyase with its substrates: L-tyrosine and L-serine, and the competitive inhibitors: L-alanine, L-phenylalanine, L-m-tyrosine, were studied. It was demonstrated that the enzyme catalyzed a half-transamination reaction between substrates or inhibitors and the protein-bound pyridoxal phosphate. The products of this side-reaction, pyridoxamine phosphate and the respective keto acids, were identified. The kinetic parameters were determined for beta-elimination of L-tyrosine and of L-serine, and for the transamination of L-serine and the inhibitors used. The transfer of the amino group to the coenzyme takes place in the direction from amino acid to pyridoxal phosphate, but not in the opposite direction, i.e. the transamination is irreversible.

Citrobacter↗