Thiosulfinates: Cytotoxic and Antitumor Activity.

Pharmacological value of some natural compounds makes them attractive for use in oncology. The sulfur-containing thiosulfinates found in plants of the genus Allium have long been known as compounds with various therapeutic properties, including antitumor. Over the last few years, the effect of thiosulfinates on various stages of carcinogenesis has been actively investigated. In vitro and in vivo studies have shown that thiosulfinates inhibit proliferation of cancer cells, as well as they induce apoptosis. The purpose of this review is to summarize current data on the use of natural and synthetic thiosulfinates in cancer therapy. Antitumor mechanisms and molecular targets of these promising compounds are discussed. A significant part of the review is devoted to consideration of a new strategy for treatment of oncological diseases - use of the directed enzyme prodrug therapy approach aiming to obtain antitumor thiosulfinates in situ.

O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site.

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.

Analyses of pre-steady-state kinetics and isotope effects of the γ-elimination reaction catalyzed by Citrobacter freundii methionine γ-lyase.

Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5'-phosphate-dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and ß-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.

Kinetic and pharmacokinetic characteristics of therapeutic methinoninе γ-lyase encapsulated in polyion complex vesicles.

Therapeutic enzymes used for the treatment of a wide range of human disorders often suffer from suboptimal pharmacokinetics and stability. Engineering approaches such as encapsulation in micro- and nanocarriers, and replacements of amino acid residues of the native enzyme provide significant potential for improving the performance of enzyme therapy. Here, we develop a nanodelivery system on the base of polyion complex vesicles (PICsomes) that includes methionine γ-lyase (MGL) as a therapeutic enzyme. We have two strategies for using the enzyme: first, methionine γ-lyase is an anticancer agent removing l-methionine from plasma, second, the binary system methionine γ-lyase/S-alk(en)yl-l-cysteine sulfoxides is effective in enzyme prodrug therapy (EPT). Various lengths polymers were synthesized, and two mutant forms of the enzyme were used. The catalytic and pharmacokinetic parameters of the nanoformulations were investigated. The catalytic efficiencies of encapsulated enzymes were comparable to that of native enzymes. Pharmacokinetic analysis has shown that inclusion into PICsomes increases half-life of the enzymes, and they can be safely administered in vivo. The results suggest the further use of encapsulated MGLs for EPT and anticancer therapy, and this strategy could be leveraged to improve the efficiency of enzyme-based therapies for managing serious human diseases.

O-acetylhomoserine sulfhydrylase from Clostridium novyi. Cloning, expression of the gene and characterization of the enzyme.

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l-methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.

Methionine γ-lyase in enzyme prodrug therapy: An improvement of pharmacokinetic parameters of the enzyme.

Citrobacter freundii methionine γ-lyase (MGL), in addition to the physiological reaction, catalyzes the ß-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates, which have antibacterial activity. We have obtained the mutant form C115H MGL, which cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme does. The binary system MGL/S-alk(en)yl-L-cysteine sulfoxides may be considered as a new pharmacological pair in enzyme prodrug therapy (EPT). Despite of the successful application of this pair in antibacterial studies in vitro, in vivo experiments may lead to several problems typical of therapeutic proteins including a relatively short-lasting biological activity. To circumvent these problems, we have investigated several approaches to improve safety and efficacy of the enzyme component of the pharmacological pair. This included covalent attachment of poly(ethylene glycol) to the enzyme, its encapsulation in liposomes and polymeric vesicles (PICsomes). The steady-state and pharmacokinetic parameters of modified/encapsulated enzyme were determined. It was demonstrated that the encapsulation in PICsomes prolongs in vivo stability of C115H MGL to over 42 h compared to PEGylated enzyme (3 h). Antibacterial activity of binary system ("pharmacological pair") modified/encapsulated enzyme/S-alk(en)yl-L-cysteine sulfoxides was tested and remained the same as for the naked enzyme. Thus, the usage of MGL-loaded PICsomes as enzymatic nanoreactors in ETP to produce antimicrobial thiosulfinates is promising.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Serine 51 residue of Citrobacter freundii tyrosine phenol-lyase assists in C-α-proton abstraction and transfer in the reaction with substrate.

In the spatial structure of tyrosine phenol-lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l-tyrosine and 3-fluoro-l-tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4'-position of the leaving phenol group in the reaction with the natural substrate.

Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l-norleucine.

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.

Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the ß-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates.

Antimicrobial activity of thiosulfinates in situ produced by mixtures of Citrobacter freundii methionine γ-lyase (MGL) with new substrates, l-methionine and S-(alkyl/allyl)-l-cysteine sulfoxides has been recently demonstrated (Anufrieva et al., 2015). This opens a way to the rational design of a new biotechnologically relevant antimicrobial drug producer. To increase the efficiency of the enzyme toward sulfoxides, the mutant forms of MGL, with the replacements of active site cysteine 115 with alanine (C115A MGL) and histidine (C115H MGL) were obtained. The replacement of cysteine 115 by histidine results in the loss of activity of the mutant enzyme in the γ-elimination reaction of physiological substrate, whereas the activity in the ß-elimination reaction of characteristic substrates persists. However, the catalytic efficiency of C115H MGL in the ß-elimination reaction of S-substituted l-cysteine sulfoxides is increased by about an order of magnitude compared to the wild type MGL. The antibacterial activity of C115H MGL mixtures with a number of sulfoxides was assessed against Gram-positive and Gram-negative bacteria. The bacteriostatic effect was more pronounced against Gram-positive than against Gram-negative bacteria, while antibacterial potential proved to be quite similar. Thus, the mutant enzyme C115H MGL is an effective catalyst, in particular, for decomposition of sulfoxides and the pharmacological couples of the mutant form with sulfoxides might be new antimicrobial agents.

The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and ß-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-ß- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and ß-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors.

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the ß-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

Alliin is a suicide substrate of Citrobacter freundii methionine γ-lyase: structural bases of inactivation of the enzyme.

The interaction of Citrobacter freundii methionine γ-lyase (MGL) and the mutant form in which Cys115 is replaced by Ala (MGL C115A) with the nonprotein amino acid (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) was investigated. It was found that MGL catalyzes the ß-elimination reaction of alliin to form 2-propenethiosulfinate (allicin), pyruvate and ammonia. The ß-elimination reaction of alliin is followed by the inactivation and modification of SH groups of the wild-type and mutant enzymes. Three-dimensional structures of inactivated wild-type MGL (iMGL wild type) and a C115A mutant form (iMGL C115A) were determined at 1.85 and 1.45 Šresolution and allowed the identification of the SH groups that were oxidized by allicin. On this basis, the mechanism of the inactivation of MGL by alliin, a new suicide substrate of MGL, is proposed.

A straightforward kinetic evidence for coexistence of "induced fit" and "selected fit" in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris.

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme ("induced fit") is realized in the case of mutant Y72F enzyme reaction with OIA. For the reaction of mutant enzyme with l-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates ("selected fit"). For the reaction with S-ethyl-l-cysteine the observed concentration dependence of kobs agrees with the realization of both kinetic schemes, the "selected fit" becoming predominant at lower concentrations of substrate, the "induced fit"- at higher ones. In the reaction with S-ethyl-l-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic α,ß-elimination of ethylthiol from S-ethyl-l-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.

The role of substrate strain in the mechanism of the carbon-carbon lyases.

The carbon-carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from l-tryptophan and l-tyrosine, respectively. These PLP-dependent enzymes show high sequence homology (∼40% identity) and both form homotetrameric structures. Steady state kinetic studies with both enzymes show that an active site base is essential for activity, and α-deuterated substrates exhibit modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500nm. In the presence of phenol and indole analogues, 4-hydroxypyridine and benzimidazole, the quinonoid intermediates of TPL and TIL decay to aminoacrylate intermediates, with λmax at about 340nm. Surprisingly, there are significant kinetic isotope effects on both formation and subsequent decay of the quinonoid intermediates when α-deuterated substrates are used. The crystal structure of TPL with a bound competitive inhibitor, 4-hydroxyphenylpropionate, identified several essential catalytic residues: Tyr-71, Thr-124, Arg-381, and Phe-448. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg-381(TPL)/Ile-396 (TIL); Thr-124 (TPL)/Asp-137 (TIL), and Phe-448 (TPL)/His-463 (TIL). Mutagenesis of these residues results in dramatic decreases in catalytic activity without changing substrate specificity. The conserved tyrosine, Tyr-71 (TPL)/Tyr-74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. Mutation of Arg-381 and Thr-124 of TPL to alanine results in very low but measurable catalytic activity. Crystallography of Y71F and F448H TPL with 3-fluoro-l-tyrosine bound demonstrated that there are two quinonoid structures, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cß-Cγ bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cß-Cγ bond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg-381 and the OH of Thr-124, and the phenyl rings of Phe-448 and 449 provide steric strain. Based on the effects of mutagenesis, the substrate strain is estimated to contribute about 10(8) to TPL catalysis. Thus, the mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL.

Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and ß-protons of competitive inhibitors.

The three-dimensional structure of the external aldimine of Citrobacter freundii methionine γ-lyase with competitive inhibitor glycine has been determined at 2.45 Å resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Cα-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Cα-proton to C4'-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Cα-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Cα-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Cα-atom of glycine to C4'-atom of the cofactor. The exchange of Cα-pro-(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of ß-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.

Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C-C bond cleavage.

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cß-Cγ bond of L-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon-carbon bond. As for most other pyridoxal 5'-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-L-tyrosine followed by flash-cooling. The X-ray structures, determined at ~2.0 Å resolution, reveal two quinonoid geometries: "relaxed" in the open and "tense" in the closed state of the active site. The "tense" state is characterized by changes in enzyme contacts made with the substrate's phenolic moiety, which result in significantly strained conformation at Cß and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate's Cß-Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme-ligand contacts remain almost exactly the same as in the "tense" quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.