Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)].poly[d(AT)], poly(dA).poly(dT), and DNA dodecamer with the sequence 5'-CGTATATATACG-3'. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)].poly[d(AT)] and poly(dA).poly(dT).

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-β-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 β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-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.

The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 – dependent methionine γ-lyase, which metabolizes it in the patient’s body. The enzyme catalyzes the γ- and β-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and β-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.

Spectral properties of the internal Schiff base in tyrosine phenol-lyase have been investigated in the presence of an activating cation K 1 and a cation-inhibitor Na 1 . The holoenzyme absorption spectra in the pH range 6.5±8.7 were recorded in the presence of K 1 . No apparent pK a 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 H -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 1 with Na 1 . This replacement leads to a significant decrease in the pK a value of pyridinium nitrogen of the pyridoxal-P.Keywords: tyrosine phenol-lyase; internal Schiff base; monovalent cations; ionic forms; tautomeric forms.Bacterial tyrosine phenol-lyase (EC 4.1.99.2) is a pyridoxal-P-dependent enzyme that catalyses b-elimination reaction of l-tyrosine to give phenol, pyruvic acid, and ammonia (see below). The enzyme is found mainly in the Enterobacteriaceae family [1]. The enzyme from Citrobacter freundii is the best studied. The tyrosine phenol-lyase gene from this bacterium was cloned and the protein amino-acid sequence was deduced from cDNA structure. The molecule of the enzyme consists of four identical polypeptide chains of 51.4 kDa each [2]. X-ray studies of the C. freundii apoenzyme [2] and C. freundii and Erwinia herbicola holoenzymes [3,4] have shown that the tetramer consists of two catalytical dimers, each of which contains an active site formed by the residues of two subunits. In each subunit, the :-amino group of lysine residue (Lys257) forms an aldimine bond (the internal Schiff base, the internal aldimine) with the coenzyme aldehyde group.Tyrosine phenol-lyase requires monovalent cations for its activity. Most monovalent cations are enzyme activators, whereas Na 1 is inhibitor [5]. The monovalent cationactivator (one in each subunit) is bound on the border between the catalytic dimer subunits with a co-ordination number of 7 [3,4].The first step of various reactions of amino-acid metabolism catalysed by pyridoxal-P-dependent enzymes is the formation of the external Schiff base (the external aldimine) as a result of transaldimination between the internal aldimine and the aminoacid substrate. Knowledge of ionic and tautomeric forms of the internal Schiff base is necessary for understanding of the mechanism of transaldimination step. To achieve this, we have studied spectroscopic properties of the internal aldimine of recombinant tyrosine phenol-lyase from C. freundii in the presence of K 1 and Na 1 cations. M A T E R I A L S A N D M E T H O D S MaterialsLactate...

No abstract

No abstract

No abstract