Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes

Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.     

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.     

Purification,characterization and identification of tryptophan aminotransferase from rat brain

Tryptophan aminotransferase was purified from rat brain extracts. The purified enzyme had an isoelectric point at pH 6.2 and a pH optimum near 8.0. On electrophoresis the enzyme migrated to the anode. The enzyme was active with oxaloacetate or 2-oxoglutarate as amino acceptor but not with pyruvate, and utilized various L-amino acids as amino donors. With 2-oxoglutarate, the order of effectiveness of the L-amino acids was aspartate > 5-hydroxytryptophan > tryptophan > tyrosine > phenylalanine. Aminotransferase activity of the enzyme towards tryptophan was inhibited by L-glutamate. Sucrose density gradient centrifugation gave a molecular weight of approx. 55,000. The enzyme was present in both the cytosol and synaptosomal cytosol, but not in the mitochondria. The isoelectric focusing profile of tryptophan: oxaloacetate aminotransferase activity was identical with that of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) activity, with both subcellular fractions. On the basis of these data, it is suggested that the enzyme is identical with the cytosol aspartate: 2-oxoglutarate aminotransferase.     

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Aspartate aminotransferase of Pediococcus cerevisiae.

A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity.     

The enantiomeric error frequency of aspartate aminotransferase

The enantiomeric error frequency of aspartate aminotransferase (mitochondrial isoenzyme from chicken) was assessed by adding the enzyme in high concentration (0.89 mM) to a mixture of L-glutamate and 2-oxoglutarate (12 and 1.2 mM, respectively, at pH 7.5 and 25 degrees C). The substrates continuously undergo the transamination cycle under these conditions. Thereby, L-glutamate is progressively racemized, a 1:1 ratio of two enantiomers being reached within 240 h. The enantiomeric error frequency, i.e. the ratio of the rate of D-glutamate production and the rate of the transamination reaction with glutamate and 2-oxoglutarate as substrates, is 1.5 x 10(-7). D-Glutamate is also converted to a 1:1 racemic mixture. The racemizing activity of a mixture of free pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate is about two orders of magnitude lower than that of aspartate aminotransferase. The error frequency of the enzyme in the case of the C4 substrate pair aspartate and oxalacetate is 3.4 x 10(-8), i.e. 4 times lower than that with the C5 substrate pair.     

Aspartate aminotransferase isozymes from rabbit liver. Purification and properties

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.     

Separation and kinetic properties of the molecular forms of chicken liver cytoplasmic aspartate aminotransferase

A method is proposed for the separation of the five molecular forms, alpha, beta, gamma, delta and epsilon, of chicken liver cytoplasmic aspartate aminotransferase free from lactate dehydrogenase activity. These molecular forms varied in isoelectric point, but no differences were observed either in their Michaelis constants or in the degree of their inhibition by excess of 2-oxoglutarate or L-aspartate.     

The use of steady-state rate equations to analyse progress curve data.

Analysis of progress curves for enzyme-catalyzed reactions has been made by using a procedure that does not require the derivation of complex integrated rate equations. The method involves conversion of progress curve data to reaction velocities that are then fitted to the appropriate differential rate equation. Application of the procedure to data obtained for the reaction catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), showed that the resulting values for the kinetic parameters agreed well with those obtained by conventional progress curve analysis (Duggleby, R.G. and Morrison, J.F. (1978) Biochim. Biophys. Acta 526, 398--409).     

INHIBITION OF ALANINE AND ASPARTATE AMINOTRANSFERASES BY α-OXODERIVATIVES OF THE BRANCHED-CHAIN AMINO ACIDS

Abstract—

• 1 L-Alanine: α-oxoglutarate aminotransferase was partly purified from rat brain and liver. The enzyme from the brain has about 10 times less activity than that from the liver.
• 2 Both enzymes have identical apparent K_m values for L-alanine, L-glutamate, α-oxoglutarate and pyruvate. Moreover they are competitively inhibited by L-leucine. α-oxoisocaproate and α-oxotsovalerate. Obtained K, values are very similar and do not depend on the course of reaction.
• 3 α-Oxoisocaproate inhibits the activity of crystalline L-aspartate: α-oxoglutarate aminotransferase; Kj is about 4–7 mM.
• 4 The pyridoxamine form of L-alanine: α-oxoglutarate aminotransferase seems to be more sensitive to the inhibitory effect of the compounds investigated.
• 5 The effect of branched-chain amino acids and their α-oxoanalogues on the metabolism of amino groups in maple syrup urine disease is discussed.

     

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.     

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.     

The transport of L-cysteinesulfinate in rat liver mitochondria.

1. The mechanism of L-cysteinesulfinate permeation into rat liver mitochondria has been investigated. 2. Mitochondria do not swell in ammonium or potassium salts of L-cysteinesulfinate in all the conditions tested, including the presence of valinomycin and/or carbonylcyanide p-trifluoromethoxyphenylhydrazone. 3. The activation of malate oxidation by L-cysteinesulfinate is abolished by aminooxyacetate, an inhibitor of the intramitochondrial aspartate aminotransferase, it is not inhibited by high concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (in contrast to the oxidation of malate plus glutamate) and it is decreased on lowering the pH of the medium. 4. All the aspartate formed during the oxidation of malate plus L-cysteinesulfinate is exported into the extramitochondrial space. 5. Homocysteinesulfinate, cysteate and homocysteate, which are all good substrates of the mitochondrial aspartate aminotransferase, are unable to activate the oxidation of malate. Homocysteinesulfinate and homocysteate have no inhibitory effect on the L-cysteinesulfinate-induced respiration, whereas cysteate inhibits it competitively with respect to L-cysteinesulfinate. 6. In contrast to D-aspartate, D-cysteinesulfinate and D-glutamate, L-aspartate inhibits the oxidation of malate plus L-cysteinesulfinate in a competitive way with respect to L-cysteinesulfinate. Vice versa, L-cysteinesulfinate inhibits the influx of L-aspartate. 7. Externally added L-cysteinesulfinate elicits efflux of intramitochondrial L-aspartate or L-glutamate. The cysteinesulfinate analogues homocysteinesulfinate, cysteate and homocysteate and the D-stereoisomers of cysteinesulfinate, aspartate and glutamate do not cause a significant release of internal glutamate or aspartate, indicating a high degree of specificity of the exchange reactions. External L-cysteinesulfinate does not cause efflux of intramitochondrial Pi, malate, malonate, citrate, oxoglutarate, pyruvate or ADP. The L-cysteinesulfinate-aspartate and L-cysteinesulfinate-glutamate exchanges are inhibited by glisoxepide and by known substrates of the glutamate-aspartate carrier. 8. The exchange between external L-cysteinesulfinate and intramitochondrial glutamate is accompanied by translocation of protons across the mitochondrial membrane in the same direction as glutamate. The L-cysteinesulfinate-aspartate exchange, on the other hand, is not accompanied by H+ translocation. 9. The ratios delta H+/delta glutamate, delta L-cysteinesulfinate/delta glutamate and delta L-cysteinesulfinate/delta aspartate are close to unity. 10. It is concluded that L-cysteinesulfinate is transported by the glutamate-aspartate carrier of rat liver mitochondria. The present data suggest that the dissociated form of L-cysteinesulfinate exchanges with H+-compensated glutamate or with negatively charged aspartate.     

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.     

The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.

1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.     

Use of β-Methylene-D,L-Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.     

Dissociation of aspartate aminotransferase into subunits. Effect of ligands upon this dissociation.

Frontal and zonal analysis of the chromatography of aspartate aminotransferase (EC2.61.1), pig heart cytosolic enzyme, on Bio-Gel P150 shows that holo- and apoenzyme can dissociate at pH 8.3. Ultracentrifugation and fluorescence depolarization confirm this result. Kinetic analysis of the fluorescence depolarization experiments favors a biphasic phenomenon: a few minutes for the faster one and several hours for the slower one. The apparent dissociation constant is 0.8 muM for the apoenzyme and 0.18 muM for the pyridoxal 5'-phosphate form of the holoenzyme. In the presence of sucrose or 0.1 M L-aspartate or a mixture of 70 mM L-glutamate and 2 mM alpha-ketoglutarate, the holoenzyme is dimeric at concentrations higher than 5 nM. The addition of a mixture of the substrates L-glutamate and alpha-ketoglutarate to a monomeric holoenzyme leads to dimerization. The stability of the dimeric form is in the order: holoenzyme + substrates greater than apoenzyme.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.