Spectral properties of aspartate transaminase from chicken heart cytosol

For the first time an interaction between aspartate transaminase (EC 2.6.1.1.) from chicken heart cytosol and the substrates and their analogues has been investigated by means of circular dichroism and absorption spectra (at pH 5,0-8,0 range). The asymmetry factor of the native enzyme and the enzymes--substrate intermediates was found. The results obtained were explained in terms of changes of the enzyme's active site conformation.     

Study of the role of arginine residues in aspartate transaminase from chicken heart cytosol

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.     

Reactivity of the cysteine and tyrosine residues of aspartate transaminase from chicken heart cytosol

Aspartate transaminase (EC 2.6.1.1) from chicken heart cytosol contains 4 thiol groups per subunit. Two of them are fully buried. One exposed SH group is readily modified by iodoacetamide, N-ethylmaleimide, tetranitromethane, 5,5′-dithio-bis(2-nitrobenzoate), 4,4′-dipyridyl disulfide and p-mercuribenzoate. A further SH group is semi-buried: while inaccessible for alkylating reagents and disulfides, it can be blocked by p-mercuribenzoate at pH about 5 (but not at pH 8). Treatment of the enzyme with tetranitromethane in the absence of substrates leads to nitration of maximally 0.8 tyrosine residue per subunit; in the presence of amino and keto substrate 1.65 eq of nitrotyrosine is formed, with a moderate decrease of enzymic activity.     

Crystallization of free aspartate aminotransferase from chicken heart cytosol]

Homogeneous aspartate aminotransferase (purity--99%, yield--70%) has been prepared from chicken heart cytosol. The purification procedure included fractionation with ammonium sulfate and ethanol and crystallization. Crystals (0.3 x 0.5 x 2 mm) of the free enzyme were prepared from ammonium sulfate solution and studied by X-ray analysis at 2.5 A resolution.     

31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart

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.     

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.     

A proteolytic enzyme bound to the aspartate transaminase of swine heart cytosol]

Purified preparations of aspartate transaminase from pig heart cytosol contain a tightly bound proteolytic enzyme (approximately 2, 5%). The enzyme was separated from aspartate transaminase by gel-filtration on Sephadex G-100 in the presence of sodium dodecyl sulfate and by affinity chromatography on the column with Sepharose, containing covalently bound denaturated aspartate transaminase. Protease has a pH optimum of 9.0 and molecular weight of about 23.000-25.000. The proteolysis rates of different subforms of aspartate transaminase depend on their denaturation lability. A more stable choloenzyme is split at a slower rate than the apoenzyme. An enriched preparation of protease was also shown to split glutamate decarboxylase from E. coli and had no effect on cysteinlyase from hen egg, as well as on lactate dehydrogenase and albumin.     

Three-dimensional structure at 5 Å resolution of cytosolic aspartate transaminase from chicken heart

An X-ray study of orthorhombic crystals of cytosolic aspartate transaminase from chicken heart has been carried out at 5 Å resolution. The crystals belong to space group P2₁2₁2₁, with unit cell dimensions $\text{a = 62.7}\text{A}\text{?}\text{, b = 118.1}\text{A}\text{?}\text{, c = 124.5}\text{A}\text{?}$. The electron density map has been calculated on the basis of five heavy-atom derivatives. The model of the molecule derived from this map revealed clearly two subunits of similar structure related by a non-crystallographic dyad. The secondary structure of the protein comprises nine helical segments per subunit.The enzyme has been shown to be catalytically active in the crystal form. Removal of the coenzyme from the crystals made it possible to derive from the difference Fourier map the position of the active site in the enzyme molecule.Significant conformational changes have been observed which accompany the interconversion of intermediates of the enzymic reaction.     

Modification of histidine residues with diethylpyrocarbonate in aspartate transaminases from pig and chicken heart cytosol]

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.     

Aspartate transaminase from cytosol of the chicken muscles: Its purification and various properties]

Homogeneous cytosolic aspartate aminotransferase was prepared from chicken muscle. The purification procedure involves heat and ammonium sulfate fractionation, chromatography on ion-exchage cellulose CM-52 and crystallization of the enzyme. A comparison of some properties of aspartate aminotransferases from chicken skeletal muscle and heart has been made. Both enzymes were found identical in terms of their electrophoretic mobility, isoelectric point, pyridoxal phosphate content and the amount of SH-groups.     

Effect of chemical modification and carboxylate anions on transamination of phenylalanine and alanine in the active center of chicken cytosol aspartate transaminase

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.     

Linear dichroism of chicken cytosol aspartate aminotransferase oriented in polyacrylamide gel

Cytosolic chicken heart aspartate aminotransferase (EC 2.6.1.1) was incorporated in polyacrylamide gel and partially oriented by compressing the gel block in two mutually perpendicular directions. The linear dichroism (LD) was recorded in a dichrograph equipped with a quarter-wavelength device which transforms circularly polarized light into linearly polarized. Spectra were resolved with lognormal distribution curves. A marked difference has been found between reduced linear dichroism values (LD/A) in the absorption bands of the protonated (430 nm) and nonprotonated (360 nm) forms of the internal pyridoxal phosphate--lysine aldimine. This finding indicates that protonation of the internal aldimine bond induces a change in direction of the transition dipole moment within the coenzyme ring or reorientation of the ring. Formation of the external aldimine with 2-methylaspartate is accompanied by a decrease of the reduced LD value in the 430 nm band. On the other hand, binding of the dicarboxylate anions, which imitates formation of the noncovalent adsorption Michaelis complex, results in a marked increase of the reduced LD value in the 430 nm band. These data suggest that the coenzyme ring tilts in opposite directions upon noncovalent substrate binding and upon subsequent formation of the external aldimine.     

Aspartate aminotransferase from chicken heart cytosol. Characterization of SH-groups]

Homogeneous aspartate aminotransferase has been prepared from chicken heart cytosol. The purification procedure includes fractionation with NH4-sulfate and with ethanol, chromatography on ion-exchange cellulose DE-32 and on hydroxylapatite. Crystallization of the enyme is described. The enzyme was shown to contain 4 SH-groups per protein subunit of molecular weight 50 000. Two of the SH-groups are fully buried, they can be blocked with thiol reagents only upon denaturation of the protein. One exposed SH-group is readily modified at alkaline pH by iodoacetamide, N-ethymaleimide or tetranitromethane, without any inhibition of enzymic activity; this group readily reacts also with 5,5,-ditthiobis (2-nitrobenzoate) and p-mercuribenzoate. One SH-group is semi-buried: it is inaccessible to the above-mentioned reagents at pH 8, but can be blocked by p-mercuribenzoate at pH about 5. Blocking with p-mercuribenzoate of two SH-groups-the exposed and the semi-buried one-lowers enzymic activity to 70% of the initial value. Syncatalytic modication of a SH-group observed in aspartate aminotransferase from pig heart cytosol does not occur in chicken enzyme.     

Selective modification of tyrosine and cysteine residues in aspartate aminotransferase from pig heart cytosol

In the region of the active site of aspartate amino-transferase two amino acid residues — one Tyr and one Cys — are accessible to selective modification by appropriate reagents. Modification of each of the two residues singly results in certain changes of the enzyme's physico-chemical properties, but does not abolish its ability to catalyse the transamination reaction. Complete inactivation, associated with irreversible amination of the protein-bound pyridoxal-P to pyridoxamine-P, is observed only on modification of both residues.     

Syncatalytic sulfhydryl group modification in mitochondrial aspartate aminotransferase from chicken and pig heart

One sulfhydryl group of the mitochondrial isoenzyme of aspartate aminotransferase from both chicken and pig heart exhibits syncatalytic reactivity changes similar to those found previously in the cytosolic isoenzyme from pig heart (Birchmeier, W., Wilson, K.J., and Christen, P. (1973) J. Biol. Chem. $\text{248}$, 1751–1759). The reactivity of the only titratable sulfhydryl group toward 5,5′-dithiobis-(2-nitrobenzoate) is at a minimum in the free pyridoxal and pyridoxamine form of the enzyme and is increased by approximately one order of magnitude when covalent enzyme-substrate intermediates are formed. The modification of the sulfhydryl group does not affect enzymatic activity. This finding supports the earlier conclusion that the syncatalytic reactivity changes are not due to a direct participation of this group in the active site but rather to conformational adaptations of the enzyme-coenzyme-substrate compound occurring in the catalytic mechanism of aspartate aminotransferases.     

Conspicuous degree of homology between the mitochondrial aspartate aminotransferases from chicken and pig heart.

The sequence of 40 amino acid residues at the amino terminus of mitochondrial aspartate aminotransferase from chicken heart differs in only 2 positions from the sequence of mitochondrial aminotransferase of pig heart. Close structural similarity had been suggested by previous data on syncatalytic sulfhydryl modifications (Gehring H., and Christen P. (1975) Biochem. Biophys. Res. Commun. $\text{63}$, 441–447). The cytosolic aspartate aminotransferases from the same two species have now been found to differ considerably in the mode of their syncatalytic modifications. The data suggest that the cytosolic and mitochondrial aspartate aminotransferases might have evolved at different organelle-specific rates.     

Identification of the exposed and buried cysteine residues in the peptide sequence of aspartate aminotransferase from pig heart cytosol

The position of the two exposed and of one fully buried cysteine residues in the polypeptide chain of aspartate aminotransferase was established. The exposed residues are Cys-45 and Cys-82, the buried one is Cys-252. The functionally important, semiburied cysteine residue of the enzyme was previously found to be Cys-390. Available evidence indicates that the remaining fully buried cysteine residue — the one most difficultly accessible for modification — is Cys-191. Thus, the positions of all five cysteine residues of the aminotransferase molecule are identified.