[31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart]. / Issledovanie spektrov 31P-IaMR aspartat-aminotransferazy iz tsitozolia kurinykh serdets.

31P NMR spectra of the cytosolic chicken aspartate aminotransferase have been recorded at 161.7 MHz in the pH range of 5.7 to 8.2. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; difference in the chemical shift at pH 5.7 and 8.2 is only 0.35 ppm. The monoanion-dianion transition of 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein--bound coenzyme is in dianionic form throughout the investigated pH range; the small pH-dependent change of chemical shift may be due to a protein conformational change that affects O-P-O bond angle. In the presence of the 0.1 M succinate, 31P chemical shift of the enzyme remains constant in the pH range of 5.0 to 8.3.

[Effect of chemical modification and carboxylate anions on transamination of phenylalanine and alanine in the active center of chicken cytosol aspartate transaminase]. / Vliianie khimicheskoi modifikatsii i karboksilat-anionov na transaminirovanie fenilalanina i alanina v aktivnom tsentre tsitozol'noi aspartat-transaminazy kur.

Photooxidation of a histidine residue in aspartate transaminase leads to proportionate loss of the enzyme activity in reactions with L-aspartate and L-phenylalanine. Modification of two arginine residues by 1,2-cyclohexanedione strongly inhibits transamination of aspartate but, in contrast, slightly increases the rate of phenylalanine transamination. A stimulatory effect of a number of aromatic and aliphatic monocarboxylate anions on the rate of alanine transamination in the active site was observed. Indolylbutyrate was the most effective compound among those tested. Indolylbutyrate and indolylacetate act as competitive inhibitors in the case of transamination of phenylalanine or aspartate. The results were interpreted as indicating the presence in the active center of transaminase of a hydrophobic subsite participating in the binding of aromatic aminoacids.

[Improved procedure for purification of aspartate transaminase from chicken heart cytosol. Characterization of the enzyme]. / Uluchshennyi metod ochistki aspartat-transaminazy iz tsitozolia kurinykh serdets i kharakteristika preparatov fermenta.

The purification procedure reported includes fractionation of water extract from chicken hearts with ammonium sulfate, fractional precipitation with ethanol, chromatography on Whatman CM-52 cellulose and crystallization. Specific activity of the pure crystalline enzyme was 234 micromoles.min-1.mg-1, as determined in the coupled assay with malate dehydrogenase (pH 7.5; 25 degrees). The amino acid composition of the enzyme was determined and the circular dichroism spectrum was recorded in the 200-250 nm range. The spectrum shows two negative bands with extrema at 208 and 220 nm. From the circular dichroism data it is estimated that aspartate transaminase contains approximately 40% alpha-helix and 10% beta-structure.

[Study of the role of arginine residues in aspartate transaminase from chicken heart cytosol]. / Issledovania roli ostatkov arginina v aspartat-transaminaze iz tsitozolia serdtsa kur.

Reaction of 1,2-cyclohexanedione with chicken heart cytosolic aspartate transaminase results in loss of enzyme activity complying to first order kinetics up to 70% inactivation. The inactivation rate is markedly decreased in the presence of alpha-ketoglutarate, glutarate or alpha-methylaspartate. The number of arginine residues modified per subunit was approximately two (in enzyme preparations which retained 30% residual activity). The diketone-modified enzyme nearly completely loses affinity for alpha-methylaspartate and glutarate; in contrast, its ability to bind alpha-alanine and catalyze its transamination half-reaction with the bound coenzyme remains unimpaired. From these data it can be inferred that a functional arginine residue is the cationic binding site for the distal carboxyl group of the substrates. The transaminase apoenzyme was inactivated with cyclohexanedione at the same rate as reconstituted holoenzyme. Measurements of circular dichroism showed that the modified apoenzyme is capable to bind pyridoxal-P. No evidence was obtained for the presence of an arginine residue in the coenzyme binding site.

[Photooxidation of aspartate transaminase from chicken heart cytosol]. / Fotookislenie aspartat-transaminazy iz tsitozolia serdtsa kur.

Rose-bengal-sensitized photooxidation of aspartate transaminase from chicken heart cytosol results in a loss of enzymatic activity which follow first order kinetics down to 70--75% inactivation. 0.9 Histidine, 0.9 tryptophane residues and 1.5 SH groups per enzyme subunit were found to be modified in the photooxidized transaminase, which retained 26% residual activity. Photodestruction of the coenzyme was about 16%. The rate of enzyme photoinactivation is constant in the pH range 6--8, and drastically decreases with lowering pH from 6 to 4. alpha-Ketoglutarate partially protects the holoenzyme from inactivation. The apoenzyme undergoes photoinactivation at a rate almost twice as rapid as the holoenzyme. Photooxidized apotransaminase retains affinity to pyridoxal phosphate and binds as much coenzyme as the native apoenzyme. Photooxidation induces no significant alterations in the circular dichroism pattern of the enzyme in the 200 to 240 nm range. However, positive circular dichroism is markedly increased in the absorption bands of aromatic amino acids (260--300 nm). The affinity of photooxidized holoenzyme for glutarate and alpha-methyl aspartate is greatly decreased. On the other hand, photooxidized enzyme retains its ability to bind alpha-alanine and to catalize the transamination half-reaction between alpha-alanine and the bound coenzyme. These findings imply that photooxidation disturbs the binding of the distal carboxyl group of dicarboxylic substrates. This may be due to a localized conformational change induced by destruction of a photoreactive histidine residue at the active site. A role of the histidine residue in transamination reaction is discussed.

[Modification of histidine residues with diethylpyrocarbonate in aspartate transaminases from pig and chicken heart cytosol]. / Modifikatsiia ostatkov gistidina v tsitorlazmaticheskikh aspartat-transaminazakh svin'i i kuritsy pri pomoshchi dietilpirokarbonata

One and three histidine residues react with diethylpyrocarbonate (DEP) at pH 6.5 in native aspartate transminases from cffect on the enzyme activity. The rest histidine residues in aspartate transaminases (approximately 6 in the chicken enzyme and 5 in the pig enzyme) are DEP-nonreactive and can be carbetoxylated only after protein denaturation. The presence of substrates does not affect the histidine modification in transaminases.

[Interaction of aspartate transaminase from chicken heart cytosol with pyridoxal phosphate analogs]. / Vzaimodeistvie aspartat-transaminazy iz tsitozolia serdtsa kur s analogami piridoksal'fosfata

The interactions were studied of the apoenzyme of aspartate aminotransferase from chicken heart cytosol with a variety of pyridoxal-P analogues. 2-Norpyridoxal-P, 2'-n-propylpyridoxal-P, 2'-isopropylpyridoxal-P, 6-methylpyridoxal-P, and 5'-methylpyridoxal-P were shown to display coenzyme activity. Estimated relative Vmax values of the complexes of apoenzyme with the above--mentioned analogues amounted respectively to 0.8; 0,2; 0,1; 0.1 and 0.1 (taking the Vmax value of the native holoenzyme as equal 1.0). The pH-dependence of reactivation rates of the apoenzyme with pyridoxal-P and pyridoxamine-P was evaluated. 3-Deoxypyridoxal-P, 3-0-methylpyridoxal-P, 2'-phenylpyridoxal-P, 5-nor-5-beta-carboxyvinylpyridoxal and 5-nor-5-beta-carboxyethylpyridoxal fail to activate the apoenzyme, but inhibit competitively the binding of pyridoxal-P to the protein; the estimated Ki values for these analoges were 2.4-10- minus 6; 3.1-10- minus 6; 3.5-10- minus 6; 7.2-10- minus 6 and 8.3-10- minus 6 M, respectively. It is of interest to compare reactivation effects of pyridoxal-P analogues for the apoenzymes of aspartate aminotransferases from chicken and from pig heart cytosol. Although the observed effects were fairly similar, it should be noted that the relative catalytic efficiencies of complexes of the chicken apoenzyme with pyridoxal-P analogues were much lower than those of complexes formed with the pig heart apoenzyme. It thus appears that of the two enzymes tested, the chicken heart aminotransferase makes more stringent demands with respect to structure of the coenzyme.