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.

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.

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.

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.

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

The mechanism of alpha-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase. What is the role of quinonoid intermediates?

To shed light on the mechanism of isotopic exchange of alpha-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their alpha-deuterated analogues in D2O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the alpha-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of alpha-proton exchange.