Citrobacter freundii Methionine γ-Lyase: The Role of Serine 339 in the Catalysis of γ- and ß-Elimination Reactions.

Serine 339 of the active site of Citrobacter freundii methionine γ-lyase (MGL) is a conserved amino acid in most pyridoxal 5'-phosphate-dependent enzymes of the cystathionine ß-lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed γ-elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the γ- and ß-elimination reactions by the mutant form of the enzyme. The exchange rates of the C-α- and C-ß-protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 E resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of γ- and ß-elimination reactions at the stage of C-α-proton abstraction from the external aldimine, the γ-elimination reaction at the stages of coenzyme C4'-atom protonation, and C-ß-proton abstraction from a ketimine intermediate.

The Catalytic Mechanisms of the Reactions between Tryptophan Indole-Lyase and Nonstandard Substrates: The Role of the Ionic State of the Catalytic Group Accepting the Cα Proton of the Substrate.

In the reaction between tryptophan indole-lyase (TIL) and a substrate containing a bad leaving group (L-serine), general acid catalysis is required for the group's elimination. During this stage, the proton originally bound to the Cα atom of the substrate is transferred to the leaving group, which is eliminated as a water molecule. As a result, the basic group that had accepted the Cα proton at the previous stage has to be involved in the catalytic stage following the elimination in its basic form. On the other hand, when the substrate contains a good leaving group (ß-chloro-L-alanine), general acid catalysis is not needed at the elimination stage and cannot be implemented, because there are no functional groups in enzymes whose acidity is strong enough to protonate the elimination of a base as weak as Cl- anion. Consequently, the group that had accepted the Cα proton does not lose its additional proton during the elimination stage and should take part in the subsequent stage in its acidic (not basic) form. To shed light on the mechanistic consequences of the changes in the ionic state of this group, we have considered the pH dependencies of the main kinetic parameters for the reactions of TIL with L-serine and ß-chloro-L-alanine and the kinetic isotope effects brought about by replacement of the ordinary water used as a solvent with 2H2O. We have found that in the reaction between TIL and ß-chloro-L-alanine, the aminoacrylate hydrolysis stage is sensitive to the solvent isotope effect, while in the reaction with L-serine it is not. We have concluded that in the first reaction, the functional group containing an additional proton fulfills a definite catalytic function, whereas in the reaction with L-serine, when the additional proton is absent, the mechanism of hydrolysis of the aminoacrylate intermediate should be fundamentally different. Possible mechanisms were considered.

Kinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids.

Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-ß-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their ß-protons. Neither stereoselectivity in the ß-proton exchange nor noticeable α-isotope effect on the exchange rates of ß-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the rate-determining stage of the enzymatic reaction.

[Spatial structure and mechanism of tyrosine phenol-lyase and tryptophan indole-lyase].

The bacterial tyrosine phenol-lyase (EC 4.1.99.2) and tryptoptophan indole-lyase (EC 4.1.99.1) belong to pyridoxal-5'-phosphate dependent beta-eliminating lyases, catalysing the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole correspondingly. Data on the three dimentional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes, modeling distinct reaction stages of the beta-elimination of L-tyrosine are described in the paper and structural bases of monovalent cations influence of activity of the enzymes are discussed. The spectral and catalytic properties of the mutant enzymes were studied. The data thus obtained have allowed us to elucidate the catalytic functions of a number of amino acid residues and conclude that the acid-base properties of the catalytic groups of the enzymes under the optimal for the catalysis conditions in hydrophobic active sites of tyrosine phenol-lyase and tryptoptophan indol-lyase are different from those in water solutions. Study of the mechanisms of labilization of Calpha-proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process has demonstrated that in certain cases concerted reaction pathways are realized instead of stepwise ones.

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.

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.

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.

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.

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.

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.

The catalytic mechanism of tyrosine phenol-lyase from Erwinia herbicola: the effect of substrate structure on pH-dependence of kinetic parameters in the reactions with ring-substituted tyrosines.

Apparently homogeneous tyrosine phenol-lyase (TPL) from Erwinia herbicola has been prepared by a new method. The pH-dependencies of the main kinetic parameters for the reactions of Erwinia TPL with tyrosine, 2-fluorotyrosine, 3-fluorotyrosine, 2-chlorotyrosine, and 3,4-dihydroxyphenylalanine (DOPA) have been studied. The pattern of pH-dependence of V(max) depends on the nature of the substituent in the aromatic ring. For the substrates bearing small substituents (H, 2-F, 3-F) V(max) values were found to be pH-independent. For 2-chlorotyrosine and DOPA V(max) decreased at lower pH, the effect being described by equation with one pKa. Generally two bases are reflected in the pH dependence of V(max)/Km. The first base, probably is responsible for the abstraction of alpha-proton, while the second one, interacts with the phenolic hydroxyl at the stage of binding. The reaction of TPL with DOPA differs from the reactions with other tyrosines by the requirement of an additional base which is reflected in the pH-profiles of both V(max) and V(max)/Km. For the reaction of TPL from Citrobacter intermedius with DOPA only V(max)/Km values could be determined. The activity of Citrobacter enzyme towards DOPA is considerably less than that of E. herbicola enzyme, and its maximal value is attained at higher pH.

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.

2-Methyl-L-tryptophan is a substrate of tryptophanase.

Tryptophanase was generally considered to be inactive towards tryptophan derivatives substituted at 2-position of the indole ring. We have shown that cells containing tryptophanase catalyze the formation of 2-methyl-L-tryptophan from 2-methylindole and L-serine, and from 2-methylindole, pyruvate and ammonium ion. The kinetics of pyruvate formation from 2-methyl-L-tryptophan and its alpha-deuterated analogue catalyzed by homogeneous tryptophanase were examined. The primary deuterium isotope effect (kH/kD = 4.0) as well as the absorption spectrum of tryptophanase complex with 2-methyl-L-tryptophan indicate that the rate of enzymatic reaction of 2-methyl-L-tryptophan is in a considerable degree determined by the stage of removal of alpha-proton.

Tryptophanase from Escherichia coli: catalytic and spectral properties in water-organic solvents.

In water-methanol and water-dimethylformamide (DMF) (1:1 v/v) solutions tryptophanase from E.coli retains its abilities to form a quinonoid complex with quasisubstrates and to catalyze the decomposition of S-o-nitrophenyl-L-cysteine (SOPC). Both the KM and Vmax values decrease in water-organic media. The affinities of tryptophanase for L-alanine, L-tryptophan, oxindolyl-L-alanine and indole in aqueous methanol are decreased, the effect being stronger for the more hydrophobic substances. In a water solution tryptophanase catalizes the reaction of SOPC with indole to form L-tryptophan while in water-organic solvents only decomposition of SOPC is observed.

[Factors, determining the effectiveness of tryptophanase interaction with amino acids]. / Faktory, opredeliaiushchie éffektivnost' vzaimodeistviia triptofanazys aminokislotami.

Inhibition of tryptophanase-catalyzed decomposition of S-(o-nitrophenyl)-L-cysteine by a variety of amino acids has been investigated. For amino acids similar to the natural substrate and for those having minimal steric requirements for the side chain, the linear correlation exists between-RTlnKi and side chain hydrophobicity. L-ornithine and L-arginine are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This can be explained by an interaction between a positively charged group of the side chain of L-arginine or L-ornithine and a nucleophilic group of the active site. The comparison of affinity of tryptophanase for L-phenylalanine and L-homophenylalanine indicates that there is a special locus in the active site where aromatic groups are bound and oriented approximately parallel to the cofactor plane experiencing no steric hindrance. For a large number of amino acids the rates of the enzymic alpha-proton exchange in 2H2O are comparable with the rate of the reaction with L-tryptophan. Very low rate of alpha-proton exchange observed with L-alanine is an exception.

[Catalytic properties of E. coli tryptophanase in a 50% aqueous solution of methanol and dimethylformamide]. / Kataliticheskie svoistva triptofanazy iz E. coli v 50% vodnykh rastvorakh metanola i dimetilformamida.

Tryptophanase from E. coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions. Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion. The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases. In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed.

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.

[Interaction of tyrosine-phenol-lyase from Citrobacter intermedius with amino acids and their derivatives: factors determining the effectiveness of binding]. / Vzaimodeistvie tirozin-fenol-liazy iz Citrobacter intermedius s aminokislotami i ikh proizvodnymi: faktory, opredeliaiushchie éffektivnost' sviazyvaniia.

The inhibition by L-amino acids and their derivatives of tyrosine phenol-lyase is investigated. Tyramine, alpha-phenylethylamine and tryptamine have no detectable inhibition effect and hence are weakly bonded by an active site. The aromatic amino acid amides are competitive inhibitors but do not manifest an enzymatic isotope exchange of alpha-proton in D2O. Free amino acids however are competitive inhibitors and in the majority of cases exchange alpha-proton. The presence of COOH-group is therefore an important feature which determines the binding efficiency and causes the "active" conformation of the amino acid-PLP complex labelising alpha-proton. In the absence of functional and bulky groups in the amino acid side chain the hydrophobicity is found to be the main factor determining the binding efficiency. For these amino acids a correlation exists between-RTlnKi and side chain hydrophobicity. The amino acids bearing the bulky groups, i. e. valine, leucine and isoleucine have reduced binding efficiency. Lysine and arginine bearing positively charged functional groups possess no inhibition effect. Aspartic and glutamic acids are anomalously strong inhibitors taking into consideration low hydrophobicity of their side chains. One can assume that the electrophilic group able to interact with the terminal COO- -group of aspartic and glutamic acids is located in the active site of tyrosine phenollyase.