[Pyridoxal phosphate-dependent enzymes of sulfur amino acid metabolism]. / Pirodksal'fosfat-zavisimye fermenty metabolizma serosoderzhashchikh aminokislot.

Cystathionine beta-synthase and gamma-cystathionase, the two major enzymes of the transsulfuration pathway of methionine metabolism, are described. These enzymes are responsible for inborn errors, e.g., homocystinuria and cystathioninuria. The interaction of gamma-cystathionase with the cofactor, substrates and inhibitors of the general formula RONH2 containing structural fragments of substrates has been studied. A non-radioactive avidin-biotin system for the microdetection of gamma-cystathionase in dot blots has been developed. This system was applied for immunoscreening of a rat liver cDNA library in the prokaryotic expression vector lambda gt 11.

[Interaction of acetyl-CoA-carboxylase from the rat liver with alkylating amides of ATP and ADP]. / Issledovanie vzaimodeistviia atsetil-CoA-karboksilazy pecheni krysy s alkiliruiushchimi amidami ATP i ADP.

The interaction of 4-(N-chloroethyl-N-methylamino)-benzyl-gamma-amide ATP (I) and the corresponding beta-amide of ADP (II) with rat liver acetyl-CoA carboxylase was studied. Both analogs were shown to cause affinity modification of the enzyme. ATP and GoAS Ac protected the enzyme against inactivation. HCO3- increased the rate of carboxylase inactivation by analogs I and II (2.5- and 1.5-fold, respectively). The alkylating amides did not influence the rate of the bicarbonate-dependent [14C]-ADP-ATP exchange and inhibited the enzyme-catalyzed reaction of [14C]-CoAs Ac----CoAS Mal exchange, which testifies to the localization of the modified group in the CoAS Ac-binding site of the enzyme active center. Based on the affinity modification and analog size, it was found that the distance between the ATP- and CoAS Ac-binding sites of the enzyme active center can vary from 0.8 to 1.2 nm.

[Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme]. / Gamma-tsistationaza: vysokoéffektivnaia zhidkostnaia khromatografiia v rezhime neideal'noi gel'-fil'tratsii. Fiziko-khimicheskaia i immunokhimicheskaia kharakteristika fermenta.

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.

[Interaction of acetyl-CoA carboxylase from the rat liver with analogs of activated carbon dioxide and ATP]. / Vzaimodeistvie atsetil-CoA-karboksilazy pecheni krys s analogami aktivirovannoi uglekisloty i ATP.

The interaction of rat liver Ac-CoA-carboxylase with reactive and stable analogs of carbon dioxide and phosphoric acid mixed anhydrides--hypothetic intermediate of the enzyme reaction--has been studied. Carbamoylphosphate showed substrate properties, whereas phosphonacetic acid and beta-oxopropyl-alpha, alpha-diphosphonate inhibited this enzyme (Ki 3.0 and 3.5 mM correspondingly). The analog of another possible intermediate in the reaction of ATP and carbon dioxide, Appp (CH2COOH) also inhibited Ac-CoA-carboxylase (Ki = 0.7 mM).

[Organophosphate analogs of biologically active compounds. XIV. Halophosphonate analogs of ATP as acetyl CoA carboxylase inhibitors]. / Fosfororganicheskie analogi biologicheski aktivnykh soedinenii. XIV. Galoidfosfonatnye analogi ATP kak ingibitory Ac CoA-karboksilazy.

Halophosphonate ATP analogues pp[CHBr]pA and p[CHBr]ppA synthesised from bromomethylenebisphosphonate and adenosine derivatives, proved to be effective competitive inhibitors of Ac-CoA-carboxylase (CE 6.4.1.2) from rat liver (Ki = 0,2 mM). The inhibitory effects of both analogues were reversible and higher than those of some other ATP analogues. Another enzyme, Ac-CoA-synthetase (CE 6.2.1.1), with a different mode of ATP-cleavage showed wider specificity to ATP-analogues than Ac-CoA-carboxylase.

[Purification of homogeneous gamma-cystathionase and study of its structure by circular dichroism]. / Poluchenie gomogennoii gamma-tsistationazy i izuchenie ee struktury metodom krugovogo dikhroizma.

Rat liver gamma-cystathionase has been purified to homogeneity (verified by SDS electrophoresis and ultracentrifugation). The secondary and tertiary structures of the enzyme were studied by circular dichroism spectra. Our studies revealed that the holoenzyme molecule comprises approximately 22% of alpha-helices, 14% of beta-structure, 14% of beta-bends, and 50% of unordered structure. Conformational alterations of the enzyme molecule resulting from enzyme PLP elimination, reduction with sodium borohydride and irreversible inhibition by propargylglycine were examined. The enzyme's secondary structure was shown to be stable whereas the tertiary structure is labile. Saturation with PLP maintains the enzyme's optimal (catalytically active) tridimensional structure. Sodium dodecylsulfate alters its secondary (the amount of alpha-helix being raised to 34%) and tertiary structures.

[Role of beta-cyanoalanine hydratase in the synthesis of asparagine in white lupine]. / Rol' beta-tsianoalaningidratazy v sinteze asparagina u belogo liupina.

A highly active beta-cyanoalanine hydratase catalyzing the asparagine synthesis from beta-cyanoalanine was isolated from 11-day-old etyolated seedlings of white lupine (Lupinus albus) and subjected to purification. In white lupine seedlings the enzyme activity is 300 times as high as that in extracts of blue lupine (Lupinus angustifolius). The experimental data suggest that in white lupine seedlings asparagine synthesis from cysteine and cyanide occurs via beta-cyanoalanine production with its subsequent hydratation. The physiological role of beta-cyanoalanine hydratase from white lupine consists in an extensive synthesis of asparagine, whose concentration reaches 0.1 M.

[Artifactual multiple forms of beta-cyanoalanine synthase from white lupine]. / Artefaktnye mnozhestvennye formy beta-tsianolaninsintazy iz belogo liupina.

The three active forms of beta-cyanoalanine synthase (EC 4.4.1.9) from white lupine seedlings were obtained in a homogeneous state and some physico-chemical and catalytic properties of the enzyme, i.e. isoelectric points, molecular weight, amino acid composition, Km, substrate and cosubstrate specificity, etc., were studied. The three enzyme forms obtained were shown to differ insignificantly in their properties. However, their Km values for the substrates are a little higher than those for the enzyme isolated in the presence of the esterolytic protease inhibitor, namely diisopropyl fluorophosphate. A conclusion is drawn that the three active forms of beta-cyanoalanine synthase are produced under the action of proteases in the course of purification and are, accordingly, artefacts.

[Beta-cyanoalanine synthase from white lupin: some catalytic properties]. / Beta-tsianoalaninsintaza belogo liupina: nekotorye osobennosti kataliticheskikh svoistv.

Highly purified beta-cyanoalanine synthase was prepared from 11-day-old etiolated sprouts of white lupine in the presence of diisopropylfluorophosphate. The enzyme preparations were homogeneous during polyacrylamide gel electrophoresis; their specific activity exceeded that of the blue lupine enzyme 100-fold. The purification procedure included preparation of mitochondrial acetonated powder, isolation of enzyme, chromatography on DEAE-Sephadex A-50, fractionation on Sephadex G-100 and preparative polyacrylamide gel electrophoresis. Some physico-chemical and catalytic properties of the enzyme, e. g. stability upon storage, pH optimum, isoelectric point, amino acid composition, effect of buffers on the enzyme activity, substrate and cosubstrate specificity, etc., were studied. Some properties of the enzyme were found different from those of the blue lupine enzyme. The Km values for the substrates and cosubstrates of cyanoalanine synthase were determined. The effects of some anions and cations on the enzyme activity are described.

[Isolation and some properties of immobilized cystathionine-beta-synthase]. / Poluchenie i nekotorye svoistva immobilizovannoi tsistationin-beta-sintazy.

Covalent binding of the pyridoxal phosphate-dependent lyase--cystathionine-beta-synthase from chicken liver--by CNBr-activated Sepharose 4B and 6B resulted in catalytically active preparations of immobilized enzyme. Immobilized cystathionine-beta-synthase was shown to possess a higher stability as compared to the native enzyme. The maximum of activity of the obtained preparations was revealed at high temperatures (63 degrees), whereas the native enzyme had the temperature optimum at 40 degrees. The pH optimum of the enzyme activity was markedly shifted towards the alkaline region. The substrate specificity of the immobilized enzyme remained essentially unchanged.

[Steady-state kinetics of reactions catalyzed by serine sulfhydrases of Saccharomyces serevisiae]. / Statsionarnaia kinetika reaktsii, kataliziruemykh serinsul'fgidrazoi Saccharomyces cerevisiae.

The steady-state kinetics of the two substrate reaction of L-cysteine desulfation in the presence of 2-mercaptoethanol catalyzed by serine sulfhydrase from bakers yeast -- a pyridoxal phosphate-containing enzyme of the beta -- substituting lyase type -- were studied. Highly purified enzyme preparations (approximately 90% purity) of Saccharomyces cerevisiae with specific activity of 25 mumoles of H2S per 1 hr per mg of protein were used. The values of V, KS1, KS2 and alpha were calculated from the initial rates of the reaction under constant concentration of L-cysteine (S1) and variable concentration of 2-mercaptoethanol (S2) and vice versa. The data obtained suggest that under conditions of a two-substrate reaction catalyzed by serine sulfhydrase and in case of beta-cyanoalanine synthase of blue lupin the substrate binding to the enzyme is interdependent and obeys a unordered mechanism with o formation of a ternary aminosubstrate-pyridoxal phosphateenzyme-cosubstrate complex (alpha = 2.6).

[Alliinase: purification and chief physico-chemical properties]. / Alliinase: ochistka i osnovnye fiziko-khimicheskie svoistva.

A method has been developed for the purification of alliinase from garlic bulbs. High purity preparations of the enzyme were obtained with specific activity increased 67-fold over that of the homogenate. The preparations were homogeneous on electrophoresis in polyacril gel. Total activity yield was 25%. The native enzyme has a molecular weight of 130.000 and consists of two subunits. Approximately 6 moles of firmly bound pyridoxal phosphate are determined per 1 mole of the purest enzyme (4 equivalents are apparently bound non-specifically outside the active sites). The isoelectric point (pI) of alliinase in 6.2. The enzyme's absorption and circular dichroism spectra have one maximum at 430 nm, in the characteristic range of many pyridoxal-P-containing enzymes. The Km value for the natural substrate, alliin, is 5 . 10(-4) M.

[Resolution of beta-cyanoalanine synthase and recombination of its apoenzyme with pyridoxal-5'-phosphate and its analogs]. / Poluchenie apofermenta beta-tsianoalaninsintazy i reaktivirovanie ego piridoksal'-5'-fosfatom. iego analogami.

A procedure is described for the resolution of beta-cyanoalanine synthase (E.C.4.4.1.9) from blue lupine seedlings. It includes total inhibition of the enzyme the hydroxylamine (10(-2) M) followed by separation of coenzyme oxime from the protein on Sephadex G-25. Conditions for maximal apoenzyme stabilization and reactivation (nature, concentration and pH of the buffer, coenzyme concentration, etc.) were studied. The extent of apoenzyme reactivation by pyridoxal phosphate is found not to depend on the nature of the buffer (pH within the range from 7,1 to 8,8) in the preincubation medium. The interaction were investigated of cyanoalanine aposynthase with pyridoxal phosphate analogues substituted in positions 2,3,4,5 and 6 of the pyridine ring. Only 6-methyl-, 2-nor- and 5'-methyl-pyridoxal phosphate were found to activate apoenzyme to the extention 100, 56 and 31% respectively under the assay conditions. The other analogues tested do not activate apoenzyme, but they mostly interact in the enzyme's active site, competitively inhibiting the binding of pyridoxal phosphate. Ki values were determined for some analogues (according to Dixon). It is found that slight changes in structure of the coenzyme molecule markedly decrease of affinity of the analogue to cyanoalanine aposynthase. As compared with other pyridoxal-phosphate-containing enzymes, this synthase, like serine sulphhydrase and cystationine synthase, has more stringent requirement as to coenzyme structure.

[Beta-cyanoalanine synthase: Its purification and basic physico-chemical properties]. / Beta-tsianoalaninsintaza: Ochistka i osnovnye fiziko-khimicheskie svoistva

A method has been developed for the purification of beta-cyano-L-alanine synthase from etiolated 10-day-old seedlings of blue lupine. High purity preparations of the enzyme were obtained with specific activity exceeding 4000-fold that of the seedling homogenate. Preparations were homogeneous on electrophoresis in polyacrylamide gel. The yield of total activity after purification was approximately 20%. Glutamic acid is the enzyme's only N-terminal amino acid; the molecular weight of the enzyme (both native and treated with 6 M urea) is 52000. The synthase containes one mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4,8. The enzyme's absorption spectrum has a maximum at 410 nm i.e., in the characteristic range of many pyridoxal-U-containing enzymes. Data on the amino acid composition of the enzyme are presented.

[Substrate specificity of cysteine lyase]. / Substratnaia Spetsifichnost Tsisteniliazy

Substrate specificity is studied of cysteine lyase, a phosphopyridoxal-dependent enzyme belonging to the subgroup of beta-replacing lyases. This enzyme has a narrow specificity for the amino substrate; its only primary substrate is L-cysteine. Cysteine lyase has a broad specificity for the cosubstrate (replacing agent), catalysing the synthesis of L-cysteic acid from L-cysteine and sulfite ion or cystein thioesters (in the presence of some thiols). Enzyme is incapable to use alpha-phenyl- and alpha-methylcysteine as substrates. It is found that enzyme catalyses the exchange of alpha-H atoms of the aminoacid substrate cysteine with 3H2O. It does not catalyse alpha-hydrogenexchange in close structural analogues of substrate: L-alanine, D-serine, treonine, allo-threonine and 3-phosphoserine. L-Serine inhibited the synthesis of S-hydroxyethylcystein from cysteine and beta-mercaptoethanol (Ki of L-serine is 0,8-10(-2) M), participating at the first stage of reaction: the formation of a pyridoxylidenic derivative, which does not undergo the further alpha,beta-elimination of beta-replacement reactions.