Interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase.

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (Vmax = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4'-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-beta-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.

[Substrate specificity of acetyl-CoA-carboxylase from the rat liver]. / Izuchenie substratnoi spetsifichnosti atsetil-CoA-karboksilazy pecheni krysy.

The interaction between acetyl-CoA fragments and rat liver acetyl-CoA carboxylase was studied. It was found that the 3'-phosphate group did not interfere with the enzyme interaction since the substrate properties of acetyl-dephospho-CoA and acetyl-CoA are nearly identical. The non-nucleotide substrate analogs S-acetyl-pantethin and its 4'-phosphate) also displayed substrate properties (V = 1.5% and 15% of the V for acetyl-CoA carboxylation respectively). The nucleotide fragment of the acetyl-CoA molecule produced an appreciable effect on the thermodynamics of this substrate interaction with the enzyme. Its physiological role consists in all probability, in the activation and propes orientation of the acetyl group in the enzyme active center. The far more pronounced substrate properties of S-acetyl pantethin 4'-phosphate and the inhibitory properties of pantethin 4'-phosphate (compared to non-phosphorylated analogs) suggest the essential role of the beta-phosphate residue of ADP in the acetyl-CoA binding to the enzyme. The data obtained suggest also that the hydrophobic region responsible for the acyl radical binding, has a site which specifically recognizes the beta-mercaptoethyl residue of the CoA pantethin fragment. The pivotal role in the acetyl-CoA carboxylase interaction with the substrate is ascribed to the productive binding of the acetyl radical; the contribution of individual fragment of the CoA molecule is variable.

[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.

[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).

[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.

Isotopic hydrogen exchange in reactions catalysed by cysteine lyase and serine sulphhydrase.

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.