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.     

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.     

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.     

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.     

Thiol peptides from the aspartate transaminase of chicken heart cytosol

Aspartate transaminase from chicken heart cytosol was immobilized covalently on activated thiol-Sepharose and digested with trypsin. After washing, the thiol-containing peptides were eluted with 2-mercaptoethanol and further purified by gel-filtration and paper chromatography. Three pure cysteinyl peptides were isolated. One of them may be represented as Ile-(Asp, Met, Cys, Gly, Leu, Thr2)-Lys; this peptide is identical to the fragment comprizing residues 387--395 in the peptide chain of aspartate transaminase from pig heart cytosol. It thus contains a cysteine residue homologous to Cys-390 of the pig heart enzyme. The second cysteinyl peptide had the following composition and partial sequence: Tyr-Phe-Val-Ser-Glu-Gly-Phe-Glu-Leu-Phe (Cys, Ala, Glu, Ser2, Phe)Lys, which corresponds to the sequence 242--258 of the pig enzyme and thus contains a cysteine residue homologous to Cys-252. The third cysteinyl peptide was similar to the tryptic peptide of the pig enzyme containing Cys-191.     

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.     

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.     

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.     

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.     

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.     

Reaction of histidine residues in proteins with diethylpyrocarbonate: Differentlal molar absorptivities and reactivities

The applicability of the spectrophotometric determination of histidine according to Ovadi et al. [(1967) Acta Biochim. Biophys. Acad. Sci. Hung.3, 455–458] to a large range of molar ratios of diethylpyrocarbonate (DEP) to histidine requires the use of appropriate differential molar absorptivities dependent on the DEP concentration used. This improved procedure allows a more differentiated aproach to the reactivity of histidine residues in proteins.     

Study of the role of histidine residues in streptokinase molecule using modification with diethylpyrocarbonate

The interaction of streptokinase with diethylpyrocarbonate resulting in partial inactivation of the protein was studied. Eight histidine residues are blocked per streptokinase molecule by this reagent. Ethoxyformylation of streptokinase histidyls is characterized by a rate constant corresponding to modification of free L-histidine. No reactivation of streptokinase was achieved by treatment of the modified protein with hydroxylamine. The CD spectroscopy data suggest that the residues modified by diethylpyrocarbonate are of no consequence for the stabilization of the protein secondary structure. The specificity of modification of streptokinase histidine residues by diethylpyrocarbonate is discussed. Based on the gel chromatography data, it was assumed that partial inactivation of streptokinase depends on the formation of protein oligomers with a decreased activatory function.     

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.     

Modification of pig liver dimeric dihydrodiol dehydrogenase with diethylpyrocarbonate and by rose bengal-sensitized photooxidation: evidence for an active-site histidine residue.

Dihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.