Acrolein modifies and inhibits cytosolic aspartate aminotransferase

Acrolein is a reactive lipid peroxidation byproduct, which is found in ischemic tissue. We examined the effects of acrolein on cytosolic aspartate aminotransferase (cAAT), which is an enzyme that was previously shown to be inhibited by glycating agents. cAAT is thought to protect against ischemic injury. We observed that acrolein cross-linked cAAT subunits as evidenced by the presence of high molecular weight bands following SDS-PAGE. Acrolein-modified cAAT resisted thermal denaturation when compared with native cAAT. We also observed a decrease in intrinsic fluorescence (290 nm, ex; 380 nm, em). These observations are consistent with an acrolein-induced change in conformation that is more rigid and compact than native cAAT, suggesting that intramolecular cross-links occurred. Acrolein also inhibited activity, and the inhibition of enzyme activity correlated with the acrolein-induced formation of cAAT cross-links.     

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 (pK₂) 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 pK_a's of carboxylic acids in comparable solvents. Since these solvents have little effect on the pK_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.     

Induction of cytosolic aspartate aminotransferase by a high-protein diet

Induction of cytosolic aspartate aminotransferase (glutamic oxaloacetic transaminase) was observed in rat liver on administration of a high-protein diet. The enzyme activity in the liver of rats given 60% and 80% protein diet increased to 1.8- and 1.9-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of functional sGOT mRNA, measured by in vitro translation. Whereas the activity of mitochondrial aspartate aminotransferase did not increase. Induction of cytosolic aspartate aminotransferase was also detected immunocytochemically.     

The amino acid sequence of cytosolic aspartate aminotransferase from human liver.

1. The cytosolic aspartate aminotransferase was purified from human liver. 2. The isoenzyme contains four cysteine residues, only one of which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. 3. The amino acid sequence of the isoenzyme is reported, as determined from peptides produced by digestion with trypsin and with CNBr, and from sub-digestion of some of these peptides with Staphylococcus aureus V8 proteinase. 4. The isoenzyme shares 48% identity of amino acid sequence with the mitochondrial form from human heart. 5. Comparisons of the amino acid sequences of all known mammalian cytosolic aspartate aminotransferases and of the same set of mitochondrial isoenzymes are reported. The results indicate that the cytosolic isoenzymes have evolved at about 1.3 times the rate of the mitochondrial forms. 6. The time elapsed since the cytosolic and mitochondrial isoenzymes diverged from a common ancestral protein is estimated to be 860 x 10(6) years. 7. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as a Supplementary Publication SUP 50158 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.     

Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemistry of the inactivation of cytosolic aspartate aminotransferase by serine O-sulfate

The reaction of serine O-sulfate with cytosolic aspartate aminotransferase [John, R.A., & Fasella, P. (1969) Biochemistry 8, 4477] has been reinvestigated. As in the corresponding reaction with beta-chloroalanine [Morino, Y., Osman, A.M., & Okamoto, M. (1974) J. Biol. Chem. 249, 6684], the enzyme is inactivated over a 10-min period, and the absorption maximum at pH 5.4 shifts from 430 to 336 nm. Upon prolonged standing the peak shifts again over a period of 20 h to 455 nm, a behavior entirely similar to that reported by Morino et al. for beta-chloroalanine in the presence of 3 M formate. When the pH of either the 10-min product (1a) or the 20-h product (1b) is raised to 11 or above, a yellow, diffusible compound (2) is released from the protein. This compound as well as its dephosphorylation and reduction products has been isolated and studied by NMR spectroscopy. Compound 2 is identical with a compound formed from serine sulfate and glutamate decarboxylase by a similar reaction sequence [Likos, J.J., Ueno, H., Feldhaus, R.W., & Metzler, D.E. (1982) Biochemistry (preceding paper in this issue)] and is the product of an aldol condensation of pyruvate with pyridoxal phosphate. When the 20-h product 1b is reduced with sodium borohydride and then heated in a boiling water bath, a material identical with the reduction product of 2 is released. We propose that the 20-h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many enzyme-activated inhibitors of pyridoxal phosphate dependent enzymes.     

Generation process of cytosolic aspartate aminotransferase molecular forms by several treatments

-, -, and -forms of chicken liver cytosolic aspartate aminotransferase generate variants on storage (4°C, 25 days). The variants developed from each isolated form appeared as evenly spaced bands with increasing anodic mobilities after polyacrylamide gel electrophoresis (PAGE), pH 8.8, and specific staining. Their mobilities coincided with those of the more negatively charged forms present in fresh tissue. Development of faster-running variants on storage was avoided by addition of thiol reagents to the freshly isolated forms. In their presence, - and -forms were partially transformed into one and two variants with lower anodic mobilities analogous to those of native - and -forms. Short pH and heat treatments did not modify the electrophoretic patterns of the -, -, and -forms, but the incubation with 5 mMl-ascorbic acid (37C, 7h) produced more anodic active bands. The formation of these variants was inhibited by the presence, in the incubation mixture, of superoxide dismutase and catalase. The kinetic parameters of the forms submitted to the different treatments were similar to those of the freshly isolated subforms. The results obtained suggest that minor subforms of the enzyme could be generatedin vivo by a mechanism in which the oxidation of particular amino acid groups is involved.     

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide

Reaction of N-bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N-bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.     

Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis

Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.     

Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.     

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. $\text{249}$, 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.     

Kinetic studies of chloride inhibition in aspartate aminotransferase activity

The inhibitive effects of chloride anion on the activity of mitochondrial aspartate aminotransferase (L-aspartate: 2-oxoglutarate-aminotransferase EC. 2.6.1.1.) from chicken (Gallus domesticus) and turkey (Maleagris gallopavo) were studied. Steady-state velocities were obtained from a wide range of chloride concentrations. The data were fitted by rational functions of 0:2 and 1:2 for chloride, using a non-linear regression program which guaranteed the fit. The goodness of fit was improved by the use of a computer program that combined model discrimination, parameter refinement and sequential design. It was concluded that chloride aspartate aminotransferase inhibition requires a minimum velocity equation of 1:2 with regard to chloride, and a plausible kinetic mechanism for this experimental result was proposed.     

Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase

The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263). In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine. This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation. The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme. Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity. The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme. Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate. Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme. These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.     

Effect of phosphate and other inorganic anions on the activity of chicken liver cytosolic aspartate aminotransferase

Chicken liver aspartate aminotransferase was inhibited by several inorganic anions. The inhibitory effect of the anions was related to their chaotropic character. Apparent Km (2-oxoglutarate) and Km (L-aspartate) values depended on the molarity of the buffer. The profile of the curves obtained did not depend on the nature of the enzyme sample assayed. Phosphate slightly inhibited the holoaspartate aminotransferase and was a strong inhibitor of apoaspartate aminotransferase with respect to pyridoxal phosphate.     

Molecular and physiological analysis of Arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase

A single, recessive mutation in soybean (Glycine max L. Merr.), which confers a seed phenotype of increased inorganic phosphate, decreased phytic acid, and a decrease in total raffinosaccharides, has been previously disclosed (S.A. Sebastian, P.S. Kerr, R.W. Pearlstein, W.D. Hitz [2000] Soy in Animal Nutrition, pp 56-74). The genetic lesion causing the multiple changes in seed phenotype is a single base change in the third base of the codon for what is amino acid residue 396 of the mature peptide encoding a seed-expressed myo-inositol 1-phospate synthase gene. The base change causes residue 396 to change from lysine to asparagine. That amino acid change decreases the specific activity of the seed-expressed myo-inositol 1-phosphate synthase by about 90%. Radio tracer experiments indicate that the supply of myo-inositol to the reaction, which converts UDP-galactose and myo-inositol to galactinol is a controlling factor in the conversion of total carbohydrate into the raffinosaccharides in both wild-type and mutant lines. That same decrease in myo-inositol 1-phosphate synthetic capacity leads to a decreased capacity for the synthesis of myo-inositol hexaphosphate (phytic acid) and a concomitant increase in inorganic phosphate.     

Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.