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PubMed1999

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.

Aspartate Aminotransferases↗

PubMed1999

Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas.

Glutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke's solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke's solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke's solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 microM, 1 microM and 10 microM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 microM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria.

Animals↗

PubMed1988

Aminotransferase activity in the liver of diabetic mice.

Enzyme activity in the livers of mice was studied in examining the metabolic disturbances of diabetes. Spontaneously non-obese diabetic (NOD) mice, mice with alloxan-induced diabetes (Allo), and control ICR mice were used. As NOD mice undergo a spontaneous pathogenic process over time, younger and older NOD mice were compared (non-diabetic and diabetic) as were control ICR mice. Two liver enzymes became more active with age, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). AST activity increased more in the hyperglycemic mice, i.e., the diabetic NOD and the Allo mice, than in the normoglycemic group, i.e., the ICR and non-diabetic NOD mice. Abnormally high AST activity was seen in the cytosolic fraction of the liver but not in the mitochondrial fraction. The changes in enzyme activity in diabetic mice were independent of any age-associated changes. The higher AST levels in diabetic mice are thought to be consistent with their greater need for gluconeogenic substrate. AST showed a more notably higher increase than did ALT in this study, and lactate dehydrogenase showed no significant changes.

Aging↗

PubMed1986

Skeletal muscle metabolism in mice bearing adenocarcinoma. III. Activities of alanine and aspartate aminotransferases.

Activities of alanine and aspartate aminotransferases in six skeletal muscles demonstrate 114-223% and 86-146% increase respectively, over the normal by the 10th week after adenocarcinoma transplantation in mice. The white (anaerobic) muscles reveal greater increment in these two enzymes than the red (aerobic) ones. The significance of elevated aminotransferase activities in energy metabolism of the host muscles is discussed.

Adenocarcinoma↗

PubMed1999

Glutathione S-transferase Alpha 1-1 and aminotransferases in umbilical cord blood.

Recent data suggest that levels of glutathione S-transferase Alpha 1-1 in umbilical cord plasma may be a good indicator of neonatal hepatocellular integrity. In order to fully understand the significance of this new marker we compared the values of glutathione S-transferase Alpha 1-1 (GSTA1-1) with that of the well known liver function markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in arterial and corresponding venous umbilical cord blood of 93 patients. In addition, in 49 of these patients maternal venous blood was also studied. Both arterial and venous umbilical cord GSTA1-1 and AST levels were significantly higher than corresponding maternal venous levels, whereas ALT levels were not. Arterial umbilical cord GSTA1-1 correlated significantly with the corresponding AST and ALT levels (R = 0.46, P < 0.0001 and R = 0.41, P < 0.0001, respectively). Arterial umbilical cord AST correlated significantly with corresponding ALT levels (R = 0.58, P < 0.0001). Arterial umbilical cord plasma GSTA1-1 levels were significantly lower in the cesarean delivery group as compared to the vaginal birth group, whereas no difference was noted for AST or ALT. Arterial umbilical cord AST and GSTA1-1 levels correlated significantly with base deficit (R = 0.29, P = 0.005; R = 0.29, P = 0.005, respectively), whereas ALT did not (R = 0.06, P = 0.54). Arterial umbilical cord AST, ALT, and GSTA1-1 levels correlated significantly with birthweight. In conclusion, GSTA1-1 levels as assessed in neonatal umbilical cord blood, being unrelated to maternal levels, seem to be a more sensitive marker for early neonatal hepatocellular integrity as compared to ALT or AST and even might detect impaired hepatocellular integrity due to the vaginal birth process. Umbilical cord GSTA1-1 may provide a valuable indicator of neonatal condition immediately after birth, the clinical relevance of which needs to be further established.

Adult↗

PubMed2000

13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant.

Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase. The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined. The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme. The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme. For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step.

Amino Acid Substitution↗

PubMed1992

Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme.

The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.

Animals↗

PubMed1992

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.

Aspartate Aminotransferases↗

PubMed1984

Transfer of C alpha-hydrogen of glutamate to coenzyme of aspartate aminotransferase during transamination reaction.

During the transamination reaction of mitochondrial aspartate aminotransferase, transfer of tritium from the alpha-position of glutamate to the pro-S position of C4' of pyridoxamine 5'-phosphate was detected. A fast mixing and quenching device had to be used in order to reduce the number of transamination cycles undergone by the enzyme and thus to minimize the accompanying exchange of label with water. The extent of transfer of label (mean value 1.5%; range 0.8-4%) indicates that the 1,3-prototropic shift follows a stepwise rather than a concerted mechanism and that a single acid/base group is responsible for the proton transfer. The actual extent of proton transfer has to be much higher because the rate of alpha-tritium exchange with solvent was only approximately 10% of that of the turnover of unlabeled substrate, reflecting either an isotope effect or a retention of the tritium label in the reaction center during tautomerization. Under the assumption of an isotope effect, the actual transfer may be estimated to be 13%. This value is consistent with the notion of Lys-258 acting as the proton transferring group in which case the maximal value of transfer in an active site not accessible to solvent during the 1,3-prototropic shift would be 33%. However, alternative mechanisms involving Tyr-70 or a water molecule enclosed in the active site serving as acid/base group cannot be excluded on the basis of the present results. Furthermore, in these investigations aspartate aminotransferase was found to catalyze also the exchange of tritium from the beta-position of glutamate, though at a rate 350 times slower than that of the alpha-exchange.

Animals↗

PubMed1989

2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli.

The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction. The mutant enzyme contains pyridoxamine phosphate as cofactor. The structure is compared to that of the mitochondrial aspartate aminotransferase. The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140.

Aspartate Aminotransferases↗

PubMed2004

Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase.

Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.

Alanine↗

PubMed1997

Transient-state kinetics of the reaction of aspartate aminotransferase with aspartate at low pH reveals dual routes in the enzyme-substrate association process.

In aspartate aminotransferase, the coenzyme pyridoxal 5'-phosphate forms a Schiff base with the epsilon-amino group of Lys258. The pH dependency of the steady-state kinetics of the overall reaction had indirectly suggested that the Schiff-base-unprotonated form of the enzyme (EL) is the active species that binds the monoanionic form of aspartate (SH+), the predominant species of the substrate in solution. In order to obtain direct information on the association process, we carried out transient-phase kinetics of the first half-reaction of the enzyme with aspartate at various pH. The disappearance of EL (lambdamax = 358 nm) was fast and independent of pH, but the disappearance of ELH+ (Schiff-base-protonated form, lambdamax = 430 nm) was slow and dependent on pH. At pH values below 6.8 and low concentrations of aspartate, the results could be interpreted to indicate that EL reacts rapidly with SH+ to form the pyridoxamine 5'-phosphate form of the enzyme (EM), and the reaction of ELH+ proceeds via the route ELH+ right arrow over left arrow EL right arrow over left arrow EM, where the first step was found to be rate limiting from the pH jump/drop study of the enzyme. At higher pH values, the rate of disappearance of ELH+ became larger than expected from the above scheme. This deviation became apparent with increasing pH, and could be excellently explained if we consider that it is due to the reaction of ELH+ with the dianionic form of aspartate (S). Thus, the formation of the Michaelis complex of aspartate aminotransferase and aspartate can proceed via two routes; route A is the association of EL with SH+ to form EL.SH+, which converts intramolecularly to ELH+.S, and route B is the association of ELH+ with S to form ELH+.S directly. ELH+.S is the prerequisite structure for further processing of the substrate by the enzyme. The reactions of EM and oxo acids yielded almost exclusively EL and SH+, and therefore route B does not seem to play an essential role in the overall reactions of the enzyme. Route B, however, may be important in the reaction mechanisms of other pyridoxal 5'-phosphate enzymes which have only the ELH+ form.

Aspartate Aminotransferases↗

PubMed1999

Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase.

To elucidate the role of the two conserved cis-proline residues of aspartate aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195A in which the two prolines were replaced by alanine. The crystal structures of P195A and P138A/P195A have been determined at 2.3-2.1 A resolution. The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, whereas the trans conformation is adopted at the 137-138 peptide bond. Quite surprisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein. All the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913]. The kinetics of renaturation of P195A, where the alanine retains the wild-type cis conformation, is faster than wild type, whereas renaturation of P138A, which adopts the trans conformation, is slower. We conclude that cis-prolines seem to have been retained throughout the evolution of aspartate aminotransferase to possibly play a subtle role in directing the traffic of intermediates toward the unique structure of the native state, rather than to respond to the needs for a specific catalytic or functional role.

Alanine↗

PubMed1999

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate.

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.

Amino Acid Sequence↗

PubMedDate not supplied

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase.

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.

Aminobutyrates↗

PubMed1995

Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase.

Mutation of six residues of Escherichia coli aspartate aminotransferase results in substantial acquisition of the transamination properties of tyrosine amino-transferase without loss of aspartate transaminase activity. X-ray crystallographic analysis of key inhibitor complexes of the hexamutant reveals the structural basis for this substrate selectivity. It appears that tyrosine aminotransferase achieves nearly equal affinities for a wide range of amino acids by an unusual conformational switch. An active-site arginine residue either shifts its position to electrostatically interact with charged substrates or moves aside to allow access of aromatic ligands.

Arginine↗

PubMed1969

The interaction between alpha-2-macroglobulin and cationic aspartate aminotransferase.

On starch-gel or polyacrylamide-gel electrophoresis of human serum, a supernumerary zone of aspartate aminotransferase activity may be demonstrated, migrating with the slow alpha(2) protein zone. This appearance is due only to cationic aspartate aminotransferase, bound by alpha(2)-macroglobulin. The binding is strongly potentiated by dilute borate buffers.

Acrylates↗

PubMed1975

The primary structure of aspartate aminotransferase from pig heart muscle. Digestion with a proteinase having specificity for lysine residues.

Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.

Amino Acid Sequence↗