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.

[CHEMICAL AIR POLLUTION OF THE OCCUPATIONAL ENVIRONMENT AS A FACTOR FOR PROFESSIONAL RISK FOR WORKERS OF MAIN OCCUPATIONS IN THE COPPER AND NICKEL METALLURGY].

There are presented the results of hygienic researches of the harmful substances content in the air of the working area ofthe copper and nickel metallurgy. Sulfur-containing gases (primarily sulfur dioxide), to the effects of which there are exposed workers of drying, smelting, converter conversion, are shown to play a leading role among professional factors.

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.

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.

[Lipid peroxidation and thrombocyte aggregation in vibration disease]. / Sostoianie perekisnogo okisleniia i agregatsiia trombotsitov pri vibratsionnoi bolezni.

A three-fold increased level of malonic dialdehyde was found in platelets of patients suffering from the first stage of vibration disease, exposed to local vibration. The changes of the fermentative and nonfermentative lipid peroxidation (LPO) were: increased accumulation of malonic dialdehyde (by 2.5 times in ascorbate-dependent LPO and by 2.6 times in NADRH-dependent LPO). Patients with peripheral vasomotor dystonia of the upper limbs combined with vasomotor spasms of the fingers showed NADPH-dependent and ascorbate-dependent LPO respectively 41% and 29% more activated than in patients having no vasomotor spasms. In patient with vibration disease antioxidant activity was reduced by 39% on average, platelet aggregation, both spontaneous and induced by epinephrine, aggristine or adenosinediphosphoric acid, was increased. Platelet hyperaggregation in vibration disease may be caused by stimulated LPO in platelets, amassed end toxic product of LPO, decreased antioxidant activity.