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PubMed1975

Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.

Animals↗
PubMed1980

Electron density map of chicken heart cytosol aspartate transaminase at 3.5 A resolution.

Aspartate transaminase (EC 2.6.1.1., Asp-transaminase) has been studied extensively, and much is now known about its physico-chemical, catalytic and other properties. X-ray studies that can provide a structural foundation for the events that occur during the transamination reaction are under way on three species of Asp-transaminase: the cytosolic enzyme from pig and chicken hearts, and the mitochondrial chicken heart enzyme. We describe here the interpretation of an electron density map of Asp-transaminase from chicken heart cytosol at 3.5 A.

Animals↗
PubMed1966

Studies on transaminase reactions in some parasitic helminths.

By an application of Sigma-Frankel methods, two transaminase systems, glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase, were found to operate at a mesurable rate in 2 species of nematodes(Ascaris lumbricoides and Ascaridia galli), 5 species of trematodes (Clonorchis sinensis, Fasciola hepatica, Eurytrema pancreaticum, Paramphistomum cervi and Paragonimus westermani) and 5 kinds of cestodes (Diphyllobothrium mansoni, Dipylidium caninum, Taenia pisiformis, Cysticercus cellulosae and Cysticercus pisiformis). A comparison was made of the transamination reactions in nematodes and those of trematodes and cestodes. And the significance of transaminase in these parasites is discussed in relation to protein synthesis and its utilization.

Journal Article↗
PubMed2005

Disproportional exaggerated aspartate transaminase is a useful prognostic parameter in late leptospirosis.

AIM: To evaluate the hepatic dysfunction in leptospirosis is usually mild and resolved eventually. However, sequential follow-up of liver biochemical data remained lacking.. METHODS: The biochemistry data and clinical symptoms of 11 sporadic patients were collected and analyzed, focusing on the impacts of leptospirosis upon liver biochemistry tests. RESULTS: The results disclosed that of the 11 cases, 5 or 45% died. The liver biochemistry data in the beginning of the disease course were only mildly elevated. Nevertheless, late exaggerated aspartate transaminase (AST) elevations were noted in three cases who finally died when compared with the typical course. Besides, significant higher AST/alanine transaminase (ALT) ratios (AARs) of the peak levels for transaminase were also noted in the cases who eventually succumbed. The mean+/-SD of AARs for the survival group and dead group were 5.65+/-2.27 (n = 5) and 1.86+/-0.64 (n = 6) respectively (P = 0.006). The ratios of the cases who finally died were all more than 3.0. Conversely, the survival group's ratios were less than 3.0. CONCLUSION: Serial follow-up of transaminase might provide evidence to predict some rare evolutions in leptospirosis. If AST elevated progressively without a concomitant change of ALT, it might indicate an acute disease course with ensuing death. Additionally, AAR is another prognostic parameter for leptospirosis. Once the value was higher than 3.0, a grave prognosis is inevitable.

Adult↗
PubMed1976

[A proteolytic enzyme bound to the aspartate transaminase of swine heart cytosol].

Purified preparations of aspartate transaminase from pig heart cytosol contain a tightly bound proteolytic enzyme (approximately 2, 5%). The enzyme was separated from aspartate transaminase by gel-filtration on Sephadex G-100 in the presence of sodium dodecyl sulfate and by affinity chromatography on the column with Sepharose, containing covalently bound denaturated aspartate transaminase. Protease has a pH optimum of 9.0 and molecular weight of about 23.000-25.000. The proteolysis rates of different subforms of aspartate transaminase depend on their denaturation lability. A more stable choloenzyme is split at a slower rate than the apoenzyme. An enriched preparation of protease was also shown to split glutamate decarboxylase from E. coli and had no effect on cysteinlyase from hen egg, as well as on lactate dehydrogenase and albumin.

Animals↗
PubMed1984

Selective tryptic cleavage of native cytoplasmic aspartate transaminase holoenzyme.

The cytoplasmic isozyme of aspartate transaminase is inactivated by trypsin due to loss of a 19-residue peptide from the NH2-terminal region. A second peptide bond at Arg-25 is then cleaved by trypsin leaving a residual core protein, transaminase 26-412. Inactivation by trypsin resembles that for the mitochondrial enzyme (Sandmeier, E., and Christen, P. (1980) J. Biol. Chem. 255, 10284-10289), yet occurs 10 times faster for the cytoplasmic isozyme. In the mitochondrial enzyme, trypsin cleavage produces equal concentrations of proteins missing the first 26 and 31 amino acids. Sequence variation in the NH2-terminal regions can explain such differences. Specifically, the mitochondrial NH2 terminus has no trypsin-susceptible residue at position 19 and is stabilized by an electrostatic interaction between Asp-15 and Arg-292, whereas position 15 is a valyl residue in the cytoplasmic enzyme. Calorimetric data reveal both a decreased transition temperature (Td) and enthalpy (delta Hd) of denaturation in transaminases 20-412 and 26-412. Interaction of substrates with the active site chromophore and differential scanning calorimetry (DSC) reveal that catalytically inactive transaminases 20-412 and 26-412 can bind amino acid substrates and produce spectroscopically detectable conversion of the pyridoxal to the pyridoxamine form of the protein. By contrast, substrate analogs only form enzymatic Michaelis-type complexes.

Amino Acid Sequence↗
PubMed1985

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase.

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.

Bacillus↗
PubMed1981

The histochemical localization of GABA-transaminase in the efferents of the striatum.

The striatum is known to contain GABA neurons projecting to the globus pallidus, entopeduncular nucleus and substantia nigra. Following lesions of striatal neurons with kainic acid, we have observed a decrease in the histochemically detectable activity of GABA-transaminase, the enzyme responsible for GABA catabolism, in both the striatum and in those areas to which it is known to send GABA projections. These results suggest that GABA-transaminase may be contained in striatal GABA neurons and thus that GABA-transaminase histochemistry may be a useful adjunct to the biochemical demonstration of GABA pathways in the brain.

4-Aminobutyrate Transaminase↗
PubMed1989

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.

Bacillus↗
PubMed1967

Turnover of rat liver tyrosine transaminase: stabilization after inhibition of protein synthesis.

Turnover of the rat liver tyrosine transaminase in vivo was measured by a label and chase procedure under conditions where the amount of enzyme undergoes no change. Half-life of the (14)C-labeled enzyme in this basal condition was found to be 1.5 +/- 0.3 hours. Inhibitors of protein synthesis (cycloheximide or puromycin) do not appreciably influence the basal enzyme level over a 5-hour period, although these drugs will block hormonal induction of this enzyme. In pulse-labeling experiments, cycloheximide blocked transaminase synthesis almost completely. The conclusion that enzyme degradation, as well as synthesis, must be blocked when protein synthesis is stopped was confirmed in experiments showing that labeled enzyme is stable in the liver of rats treated with cycloheximide The participation of a continuously synthesized polypeptide in the degradative phase of transaminase turnover is suggested.

Animals↗
PubMed1972

Nonidentity of the aspartate and the aromatic aminotransferase components of transaminase A in Escherichia coli.

Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000.

Aspartate Aminotransferases↗
PubMed1995

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).

Amino Acid Isomerases↗
PubMed1998

Characterization of the genes encoding D-amino acid transaminase and glutamate racemase, two D-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208.

In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.

Amino Acid Isomerases↗
PubMed1975

Serum transaminase levels after experimental paracetamol-induced hepatic necrosis.

The relationship between serum transaminase levels and the extent of paracetamol-induced liver necrosis has been investigated in the rat. Three methods of histological quantitation were used to assess of necrosis--arbitrary grading, point counting, and the image-analysis computer. Highly significant correlations were obtained between the three methods and all were found to be reproducible. A close correlation was found between the extent of hepatic necrosis and the serum ASAT and ALAT 24 hours after a large dose (4 g/kg) of paracetamol. Likewise, the mean grade of necrosis correlated reasonably well with the serum enzyme levels in the recovery phase at 36 and 72 hours, although the transaminase level for a given degree of necrosis was considerably lower at 72 hours than at 24 hours. These findings suggest that serum transaminase levels gives a reliable indication of the severity of hepatic necrosis if the time of ingestion of the paracetamol is known and taken into account.

Acetaminophen↗
PubMed2002

Response of regional brain glutamate transaminases of rat to aluminum in protein malnutrition.

BACKGROUND: The mechanism of aluminum-induced neurotoxicity is not clear. The involvement of glutamate in the aluminium-induced neurocomplications has been suggested. Brain glutamate levels also found to be altered in protein malnutrition. Alterations in glutamate levels as well as glutamate-alpha-decarboxylase in different regions of rat brain has been reported in response to aluminum exposure. Thus the study of glutamate metabolising enzymes in different brain regions of rats maintained on either normal or restricted protein diet may be of importance for understanding the neurotoxicity properties of aluminium. RESULTS: Dietary protein restrictions does not have an significant impact on regional aluminum content of the brain. The interaction of aluminum intoxication and protein restriction is significant in the thalamic area and the midbrain-hippocampal region in cases of glutamate oxaloacetate transaminase. In the case of glutamate pyruvate transaminase, this interaction is significant only in thalamic area. CONCLUSION: The metabolism of amino acids, as indicated by activities of specific transaminases, of brain is altered in response to aluminum exposure. These alterations are region specific and are dependent on dietary protein intake or manipulation of the brain amino acid homeostasis.

Alanine Transaminase↗
PubMed2001

Retrospective evaluation of the effect of valproate therapy on transaminase elevations in patients with hepatitis C.

STUDY OBJECTIVE: To evaluate whether a relationship exists between valproate treatment and transaminase elevations in patients who are positive for hepatitis C virus (HCV). DESIGN: Retrospective medical record review. SETTING: Veterans affairs medical center. PATIENTS: A total of 214 HCV-positive patients; 28 were treated with valproate (study group), 186 were not (control group). INTERVENTION: Demographic characteristics; valproate treatment histories; plasma concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), albumin, and bilirubin; and exposure to other potentially hepatotoxic drugs, including probable alcohol abuse, were evaluated. MEASUREMENTS AND MAIN RESULTS: Hepatotoxicity was staged by comparing maximum ALT and AST values against upper limits of normal or patients' mean elevated baseline ALT and AST values. Data were analyzed using a logistic regression model. Control patients and those with longer durations of HCV were more likely (p<0.0171 and p<0.0142, respectively) to exhibit higher stages of hepatotoxicity. More valproate-treated patients were exposed to other potential hepatotoxins at the time of peak transaminase elevations (50% vs 39%, p<0.005), whereas more control patients received two or more potential hepatotoxins (13% vs 4%, p<0.00005). CONCLUSION: Valproate treatment, either alone or in the presence of other potential hepatotoxins, does not appear to be related to increased transaminase elevations in patients with HCV

Alanine Transaminase↗
PubMed1981

Different modes of action of inhibitors of bacterial D-amino acid transaminase. A target enzyme for the design of new antibacterial agents.

D-Amino acid transaminase from Bacillus sphaericus shows a deuterium kinetic isotope effect (VH/VD) between 2 and 3 in the transamination of alpha-protio- or alpha-deuterio-D-alanine and alpha-ketoglutarate, suggesting that alpha-proton abstraction is at least partially rate-limiting for this reaction. This transaminase also catalyzes a beta-elimination reaction with substrates such as beta-fluoroalanine with no detectable deuterium isotope effect (VH/BD = 1). These results, taken together with previous work (Soper, T. S., and Manning, J. M. (1978) Biochemistry 17, 3377-3384) suggest that the rate-limiting step in the beta-elimination reaction is solvolysis of an alpha-aminoacrylate-pyridoxal-P Schiff's base intermediate. D-Cycloserine is an active site titrant of D-amino acid transaminase. Inactivation by cycloserine can be completely reversed by dialysis against pyridoxal phosphate at neutral pH. Gabaculine is also an efficient inhibitor of this enzyme and possesses some antibacterial activity. The latter two inhibitors probably act by sequestration of the coenzyme rather than by alkylation of the protein as with the beta-halo derivatives of D-alanine.

Alanine↗
PubMed1993

Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.

D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp. YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively. To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters. Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme. The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction. KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine. Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e. it optimizes catalytic efficiency and it maintains stereochemical fidelity. The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase.

Bacillus↗