Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific labeling of both the mature (mAspAT) and precursor (pmAspAT) forms of rat liver mitochondrial aspartate aminotransferase with three different spectroscopic probes (monobromotrimethylammoniobimane, N-(iodoacetylaminoethyl)-5-naphthalene-1-sulfonic acid, and N-(1-pyrenyl)maleimide) was used to assess the possible conformational consequences of the interaction of a mitochondrial precursor protein with lipid membranes by means of fluorescence spectroscopy. The three probes react with the same cysteine residue causing a partial loss of catalytic activity whose extent depends on the nature of the probe introduced. The fluorescence intensity of the attached probes decreases upon addition of substrates or substrate analogues, indicating that the modified enzymes can undergo the open-closed conformational transitions that accompany catalysis. Both unmodified and labeled precursor proteins bind to negatively charged phospholipid vesicles, whereas the mature enzyme is unable to bind. Binding to liposomes does not affect the fluorescent properties of the attached probes. However, addition of the pseudosubstrate alpha-methylaspartate to liposome-bound precursor fails to induce the characteristic conformational changes observed with the protein free in solution. Furthermore, upon binding to liposomes the precursor protein loses enzymatic activity, and the reactive cysteine residue becomes inaccessible to reaction with thiol reagents. In contrast, the presence of liposomes has no effect on the activity, cysteine reactivity, or syncatalytic conformational transitions of the mature enzyme. It appears that interaction of pmAspAT with negatively charged phospholipids prevents the protein from undergoing the conformational transitions required for catalysis, "freezing" the enzyme in a sterically hindered but open-like conformation.
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PMID:Binding to phospholipid vesicles impairs substrate-mediated conformational changes of the precursor to mitochondrial aspartate aminotransferase. 807 48

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (L-cysteine, D-cysteine, cysteamine, L-cysteine ethyl ester, L-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5'-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GPT, and not GOT, was specifically inhibited by L-cysteine and, to a lesser extent, by D-cysteine. The association reaction: PLP + apo GPT<-->holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37 degrees C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.
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PMID:The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives. 814 66

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.
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PMID:Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification. 818 43

Sodium borohydride and sodium cyanoborohydride were assessed as potential reagents for determining ligand-induced changes in accessibility to the active-site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determined in the presence of increasing concentrations of the dicarboxylate substrate analogues glutarate and maleate. The rate of reduction decreased to a limiting value which was about 40-fold lower than the equivalent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompanied binding of dicarboxylates. Allowing for this increase, the true decrease in accessibility to NaBH3CN was estimated to be approximately 400-fold. Arguments are presented in support of a proposal that the ratio of closed to open conformer of the dicarboxylate-liganded enzyme is approximately 150. The effects of increasing ligand concentration on the reactivity of Cys390 were found to take place in the same range as was observed for NaBH3CN reduction. Conversely, very much higher concentrations of the dicarboxylates were required to protect against proteolysis by trypsin. It is concluded that NaBH3CN reduction and reactivity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an additional binding mode for the dicarboxylates.
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PMID:Probes of ligand-induced conformational change in aspartate aminotransferase. 840 95

The contribution of testosterone to the nephrotoxic effects of 1,2-dichloropropane (DCP) was assessed by a series of castration and sex hormone replacement experiments on Wistar rats. The nephrotoxic action of DCP was evaluated by measuring the accumulation of organic anion and release of aspartate aminotransferase into the incubation medium using a renal cortical slice model. Our data show that sex, castration, and testosterone pretreatment are factors that influence the effect of DCP on renal cortical slices of rats Males appear to be more sensitive to nephrotoxic effects of DCP than females, male castration prevents the nephrotoxic effects of DCP, and pretreatment of females and castrated males with testosterone increases the susceptibility to DCP. In this study an attempt was made to evaluate the role of sex differences in the expression of enzymes participating in Phase I and Phase II detoxication reactions in order to explain the differences in sensitivity of the two genders to the nephrotoxic action of DCP. Our results implicate gender-specific expression of cytochrome P-450 in the kidneys as a predominant factor that determines the different susceptibilities of male and female rats to the nephrotoxic effect of DCP. We propose that the oxidation of DCP by CYP IIE1 is the first saturable and limiting step in the metabolic activation of DCP to nephrotoxic metabolites. It appears that, despite the fact that the nephrotoxic effect of DCP is determined mainly by its cysteine-conjugated metabolites, gluthathione (GSH) content and glutathione S-transferase (GST) activity in kidney are not directly related to increased androgen-related susceptibility to DCP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of sex-related differences in nephrotoxicity of 1.2-dichloropropane in rats. 857 Aug 64

Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25 degrees C (pH 7.0) results in slight inactivation (approximately 15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and alpha-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40 mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t 1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40 mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10 mM L-glutamate, pH 7.2, 25 degrees C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25 degrees C, pH 7.2) results in significant inactivation (approximately 30%). The enzyme is protected against inactivation by the presence of alpha-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1 approximately 2 mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate approximately 0.01 min-1; pH 6.0, 25 degrees C; t 1/2 max approximately 70 min). This finding suggests that 2-oxoglutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated alpha-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with alpha-ketoglutarate. The results suggest that unsaturated alpha-keto acids and their esters may be useful probes for the study of alpha-keto acid-utilizing enzymes.
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PMID:Irreversible inactivation of aspartate aminotransferase by 2-oxoglutaconic acid and its dimethyl ester. 890 17

The C-S lysis of L-cysteine conjugates is one biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolic species. Thirteen cysteine S-conjugates were synthesized in our laboratories and incubated with aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) enzymes from porcine heart tissue. The C-S lyase (CSL) activity for each enzyme-substrate combination was determined. ASAT and ALAT were shown to exhibit CSL activity and it was also demonstrated that this activity was inhibited in the presence of the pyridoxal phosphate-dependent enzyme inhibitor amino(oxyacetic acid) confirming the pyridoxal phosphate-dependent mechanism by which C-S lysis is known to take place. This finding has potentially important implications for the risk assessment of compounds which produce L-cysteine conjugates during their biotransformation.
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PMID:Novel sources of mammalian C-S lyase activity. 893 63

Pretreatment of fasted rats with aminooxyacetic acid (AOAA, 0.25 mmol kg-1, i.p.), methimazole (MTZ, 0.35 mmol kg-1, i.p.) and acivicin (AT-125, 56 mumol kg-1, i.p.) 30 min prior to a 4-h inhalation exposure to 180-200 ppm or 150-180 ppm vinylidene chloride (VDC) was used to study the role of cysteine beta-lyase, cysteine conjugate S-oxidase and gamma-glutamyltranspeptidase (gamma-GT) in VDC-induced liver and kidney toxicity. Pretreatment with AOAA reduced by 65-95% those increases in serum alanine aminotransferase (ALAT), glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) caused by exposure to 180-200 ppm VDC. This pretreatment also prevented VDC-induced increases in aspartate aminotransferase (ASAT) and N-acetyl-beta-d-glucosaminidase (NAG) activities and in the concentration of beta 2-microglobulin (beta 2-m) in 24-h urine samples. There was only a slight potentiation of VDC-induced liver and renal toxicities by MTZ given before exposure to 180-200 ppm VDC, but potentiation became significant (40-80%) when MTZ was administered before a slightly lower level of exposure (150-180 ppm). Pretreatment with AT-125 did not significantly change the liver and renal effects of exposure to 180-200 ppm VDC. These results suggest that the formation of a cysteine conjugate may be involved in the renal and liver toxicity of VDC in fasted rats.
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PMID:Role of cysteine conjugation in vinylidene chloride-induced nephrotoxicity and hepatotoxicity in fasted rats. 893 83

Long-term exposure to trichloroethylene can cause kidney cancer in experimental animals and humans. In addition, dichloroacetylene (a breakdown product of trichloroethylene) is nephrotoxic and neurotoxic. Both trichloroethylene and dichloroacetylene are metabolized in part to the corresponding cysteine S-conjugate (i.e. S-(1,2-dichlorovinyl)-L-cysteine) which is toxic. Cysteine S-conjugate beta-lyases convert S-(1,2,dichlorovinyl)-L-cysteine to pyruvate, ammonia and a reactive fragment that adds to macromolecules, depletes cellular thiols and causes lipid peroxidation. We now show that S-(1,2-dichlorovinyl)-L-cysteine and another nephrotoxic cysteine S-conjugate, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, inactive purified cytosolic aspartate aminotransferase and purified alanine aminotransferase. These cysteine S-conjugates also inactive aspartate aminotransferase in cytosolic and mitochondrial fractions of rat brain and kidney. The present results suggest that some halogenated xenobiotics may be toxic in part through their conversion to the corresponding cysteine S-conjugate which inactivates key pyridoxal 5'-phosphate-containing enzyme.
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PMID:Inactivation of brain and kidney aspartate aminotransferases by S-(1,2,-dichlorovinyl)-L-cysteine and by S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine. 894 Jun 25

The two conjugates, S-[N-(2-hydroxyethyl)carbamoylmethyl]glutathione (GSAAE), and its corresponding mercapturic derivative N-acetyl-S-[N-(2-hydroxyethyl)carbamoylmethyl]cysteine (NCySAAE) were administered to fasted Sprague-Dawley rats as putative metabolites of vinylidene chloride (VDC). Methylthioacetylaminoethanol (MAAE) was identified in the urine of GSAAE- or NCySAAE-treated rats (0.5-2.0 mmol/kg, i.p.), as well as in the urine of VDC-treated rats (0.5-2.0 mmol/kg, p.o.). The effects of VDC, GSAAE and NCySAAE on the kidney and liver were also examined using aspartate aminotransferase (ASAT). N-acetyl-beta-D-glucosaminidase (NAG) and beta 2-microglobulin (beta 2-m) as urinary parameters of nephrotoxicity, and glutamate dehydrogenase (GLDH), sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) as serum parameters of hepatotoxicity. Unlike treatment with VDC, treatment with both GSAAE and NCySAAE failed to cause kidney and liver toxicity. The results support the hypothesis that MAAE originates from the formation of GSAAE and further metabolization to NCySAAE, and that MAAE excretion does not reveal a pathway of reactive intermediates.
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PMID:Formation of GSH-derivatives as a pathway for inactive intermediates in vinylidene chloride-treated rats. 900 91


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