Cytosolic Aspartate Aminotransferase Research Articles

Apoenzyme of aspartate aminotransferase isozymes in serum and its diagnostic usefullness for hepatic diseases

Aspartate aminotransferase in the sera of normal subjects and of patients with hepatic diseases has been immunologically separated into two isoenzymes, cytosolic aspartate aminotransferase and mitochondrial aspartate aminotransferase. The activity of the isoenzymes was measured in three different buffer solutions with or without pyridoxal 5'-phosphate. To attain maximal activation, the apoenzyme of mitochondrial fraction must be preincubated with pyridoxal 5'-phosphate longer than that of the cytosolic fraction in either of the three reaction mixtures. In most sera the activity of both isoenzymes increased substantially in the presence of pyridoxal 5'-phosphate regardless of the type of buffer solutions. Both the apoenzymatic activity and the ratio of apo- to holo-enzymatic activity of each of the isoenzymes varied among samples from the patients with hepatic diseases. However, significantly high ratios of apo- to holo-enzymatic activity of both isoenzymes were observed in the patients with hepatoma in contrast with those with other hepatic diseases. These findings suggest that the simultaneous measurement of both apo- and holo-enzyme activities of aspartate aminotransferase isoenzymes may be useful in the clinical assessment of hepatic diseases.

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. 249 , 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.

Cytosolic aspartate aminotransferase: Studies on subunit interactions with the apo/holo hybrid dimer

The apo/holo hybrid dimer of cytosolic aspartate aminotransferase from pig heart was compared with the apo/apo and holo/holo homomer with respect to manifestations of subunit interactions. The hybrid dimer was isolated by iso-electric focusing from an apo enzyme preparation semi-reconstituted with pyridoxal-5′- P. The binomial distribution pattern of pyridoxal/pyridoxal homomer, apo/pyridoxal hybrid, and apo/apo homomer obtained by the iso-electric separation as well as identical rates of binding of the coenzyme to the apo/holo hybrid and the apo/apo homomer indicate random association of the coenzyme with the apo subunits. An extensive comparative exploration of the active site regions of the apo/holo hybrid containing one active site and the holo/holo homomer containing two revealed no differences with respect to the following structural and functional properties: catalytic center activity, absorption and circular dichroism spectra of the coenzyme chromophore, p K′ a values of enzyme-bound pyridoxal-5′- P, K′ m and V′ values for aspartate and 2-ketoglutarate, the dissociation constants of the complex with competitive dicarboxylic inhibitors, the absorption spectrum and the rate of decay of the intermediate with erythro-3-hydroxyaspartate. The rate of alkylation of Cys-390 with N-ethylmaleimide was found to be about 20 times faster in the apo/apo homomer than in the pyridoxal/pyridoxal homomer. The rates of modification of the apo and of the pyridoxal subunit in the hybrid are the same as those in the respective homomers. Likewise, the syncatalytic conformational changes of the holo subunit do not affect the reactivity of Cys-390 on the adjoining apo subunit. Thus, by manifold criteria, the conformational changes that accompany transamination do not reflect subunit interactions but rather are connected with the catalytic processes at functionally isolated active sites.

Solvent interactions with the active site of the pig heart cytosolic aspartate aminotransferase

Aqueous solvent interactions with the chromophoric pyridoxal phosphate prosthetic group of aspartate aminotransferase (EC 2.6.1.1) were analyzed quantitatively with ethylene glycol, glycerol, dimethylsulfoxide (DMSO), sucrose, and xylitol as cosolvents. The smaller cosolvents perturb the visible absorption and visible dichroic spectra of the free enzyme, but this solvent perturbation is not observed with the acidic enzymeglutarate complex. Addition of cosolvents caused an increase in the enzyme's affinity for glutarate. This increase in affinity resulted from an increase in the acidic dissociation constant (p K 2) of the enzyme-glutarate complex. The changes in the acidic dissociation constant of the enzyme-glutarate complex, upon addition of cosolvents, correlate well with the changes observed in the p K a 's of carboxylic acids in comparable solvents. Since these solvents have little effect on the p K a of the enzyme itself, it is concluded that the increase in affinity is due to a specific solvation effect on a carboxyl group of the enzymebound glutarate, rather than resulting from a conformational change in the protein.

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. 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.

Catalytic function of a histidyl residue in cytosolic aspartate aminotransferase-catalyzed reactions

Methylene blue-sensitized photooxidation of the cytosolic aspartate aminotransferase from pig heart yielded a preparation which was inactive in the transamination reaction with natural substrates (L-aspartate and a-keto-glutarate) but fully active in the α, β-elimination reaction with β-chloro-Lalanine as well as in the transamination reaction with L-alanine. The inactivation results from photooxidative destruction of a histidyl residue in accord with the result reported by Martinez-Carrion et al. [ J. Biol. Chem. 242, 1426 (1967)]. These results indicates that the histidyl residue does not function as a base abstracting the α-hydrogen atom of these substrates but acts as a catalytic residue involved in the interaction with the distal carboxylate of a natural dicarboxylic substrate as proposed previously [ Morino et al., J. Biol. Chem. 249, 6684 (1974)].

The apo/holo hybrid of cytosolic aspartate aminotransferase, preparation and studies on subunit interactions

The apo/holo hydrid dimer of cytosolic aspartate aminotransferase from pig heart has been isolated by isoelectric focusing from a semi-reconstituted apo enzyme preparation. Comparison of the catalytic center activity and of the optical features of the coenzyme chromophore of the hybrid containing one active center with that of the homomer containing two revealed no differences in these properties and suggests that the subunits function independently from one another. However, differential thermal inactivation studies of hybrids and of homomers showed that the subunit carrying the coenzyme markedly stabilizes the structure of the neighboring subunit. Thus, coenzyme binding elicits functionally mute conformational changes which extend to and across the subunit interface.