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)

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.
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PMID:Characterization of histidinol phosphate aminotransferase from Escherichia coli. 1268 52

The homodimeric, pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamine transaminase K/cysteine conjugate beta-lyase (GTK/beta-lyase) has been implicated in the bioactivation of chemopreventive compounds. This paper describes the first homology model of rat renal GTK/beta-lyase and its active site residues, deduced from molecular dynamics (MD) simulations of the binding mode of 13 structurally diverse cysteine S-conjugates and amino acids after Amber-parametrization of PLP. Comparison with Thermus thermophilus aspartate aminotransferase (tAAT) and Trypanosoma cruzi tyrosine aminotransferase (tTAT), used as templates for modeling GTK/beta-lyase, showed that the PLP-binding site of GTK/beta-lyase is highly conserved. Binding of the ligand alpha-carboxylate-group occurred via the conserved residues Arg(432) and Asn(219), and Asn(50) and Gly(70). Two pockets accommodated the various ligand side chains. A small pocket, located directly above PLP, was of a highly hydrophobic and aromatic character. A larger pocket, formed partly by the substrate access channel, was more hydrophilic and notably involved the salt bridge partners Glu(54) and Arg(99*) (* denotes the other subunit). Ligand-binding residues included Leu(51), Phe(71), Tyr(135), Phe(373) and Phe(312*), and pi-stacking interactions were often observed. Tyr(135) and Asn(50) were prominent in hydrogen bonding with the sulfur-atom of cysteine S-conjugates. The observed binding mode of the ligands corresponded well with their experimentally determined inhibitory potency toward GTK/beta-lyase. The current homology model thus provides a starting point for further validation of the role of active site residues in ligand-binding by means of mutagenesis studies. Ultimately, insight in the binding of ligands to GTK/beta-lyase may result in the rational design of new ligands and selective inhibitors.
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PMID:Modeling and molecular dynamics of glutamine transaminase K/cysteine conjugate beta-lyase. 1279 91

Many studies in diverse models suggest that nitric oxide (NO) may be protective against liver injury due to ischaemia-reperfusion (IR). We evaluated, in an experimental in vivo model of rat liver partial ischaemia, the effects of pretreatment by an NO donor (spermineNONOate, 5mg/kg), and exogenous cGMP (8Br-cGMP, 16 mg/kg) or an endogenous cGMP producer (ANP, 10 microg/kg), to assess their beneficial effects. After 6h of reperfusion, 8Br-cGMP completely prevented the adverse effect of Nomega-nitro-L-arginine (10mg/kg) and 8Br-cGMP alone showed a protective action on both hepatocytes (AST, -25%, LDH, -55%) and endothelial cells (plasma hyaluronic acid (HA), -30%). ANP caused a marked decrease in AST and LDH activities only after 1h of reperfusion (AST, -30%, LDH, -40%). Pretreatment with spermineNONOate prevented hepatocyte injury after 1 and 6h of reperfusion (AST, -22%, LDH, -27%). However, neither spermineNONOate nor ANP had any protective effect on endothelial cell damage. These results confirm the beneficial effect of an NO donor and strongly suggest the implication of a cGMP pathway that does not involve a blockade of inflammatory cytokines production (IL-6 generation was unaffected by 8Br-cGMP pre-treatment). In our model, 8Br-cGMP showed a greater protective effect than ANP or spermineNONOate and so might be used to prevent hepatic injury after IR. Finally, we propose a schematic representation of the different routes for the actions of NO in protecting the liver against IR damage.
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PMID:Hepatic cytoprotection by nitric oxide and the cGMP pathway after ischaemia-reperfusion in the rat. 1462 71

The aim of this study was to investigate the role of nitric oxide (NO) in hepatic ischemia-reperfusion (I/R) injury in rats. Immunohistochemistry was used to examine the protein expression of endothelial and inducible nitric oxide synthases (eNOS, iNOS) and nitrotyrosine after I/R challenges to the liver, and blood levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), hydroxyl radical and NO were measured before ischemia and after reperfusion. Ischemia was induced by occlusion of the common hepatic artery and portal vein for 40 min, followed by reperfusion for 90 min. Reperfusion of the liver induced a significant increase in the blood concentrations of AST, ALT, LDH (n = 8; P < 0.001), hydroxyl radical (n = 8; P < 0.001) and NO (n = 8; P < 0.01). The eNOS, iNOS, nitrotyrosine, SOD1 and SOD2 protein expression was also found to increase significantly after reperfusion (n = 3). Administration of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (n = 8) had a protective effect on the I/R-related injury, but the NO donor L-arginine (L-Arg) (n = 8) potentiated the damage caused by I/R. These results suggest that reperfusion of the liver induces expression of NOS, which is related to the elevation of blood NO. The increase in hydroxyl radical concentration was accompanied by an increase in antioxidant enzyme expression (SOD1 and SOD2), and an increase in nitrotyrosine expression was also observed, reflecting the increased production of NO and oxygen radicals. We concluded from the protective effect of L-NAME and the potentiation by L-Arg that NOS expression and increases in NO and hydroxyl radical production have deleterious effects on the response to I/R in the liver.
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PMID:Ischemia and reperfusion of liver induces eNOS and iNOS expression: effects of a NO donor and NOS inhibitor. 1561 29

The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.
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PMID:Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step. 1593 11

Chronic inhibition of nitric oxide (NO) synthesis is characterized by increased blood pressure accompanied with both cardiac hypertrophy as well as renal damage. We investigated whether the angiotensin-converting enzyme (ACE) inhibitor captopril can inhibit the cardiac hypertrophy and reverse the renal failure. We tested the influence of captopril on the nitrate-nitrite (NO(x)) in plasma and heart and kidney tissues. Oxidative stress, in terms of glutathione and thiobarbituric acid-reactive substances measured as malondialdehyde, was monitored examining their involvement in the cardioprotective and renoproptective actions. Three groups of Wistar rats were used: untreated group, and rats treated with the NO synthase inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) and L-NAME plus captopril (10 mg/kg/day). Systolic, diastolic and mean blood pressure (BPs, BPd and BPm respectively) was measured weekly in addition to the heart rate using rat-tail plethysmography. After 3 weeks, L-NAME significantly increased BPs, BPd and BPm. Captopril treatment reversed the increments in pressure back to normal values by the fourth week. ACE inhibition by captopril reverted the L-NAME-induced hypertrophy and inhibited the enzymatic indices of cardiac damage (glutamate oxaloacetate transaminase and lactate dehydrogenase) back to normal values. Furthermore, the NO synthesis inhibition produced renal damage as indicated by significant increase in creatinine. Captopril ameliorated the raised creatinine to normal. Chronic L-NAME treatment increased serum NO(x) levels but concomitant treatment with captopril was without effect.
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PMID:Effects of captopril on cardiac and renal damage, and metabolic alterations in the nitric oxide-deficient hypertensive rat. 1622 7

Pulmonary hypertension is prevalent in adult patients with sickle cell disease and is strongly associated with early mortality and markers of hemolysis, in particular, serum lactate dehydrogenase (LDH). Intravascular hemolysis leads to impaired bioavailability of nitric oxide (NO), mediated by NO scavenging by plasma oxyhemoglobin and by arginine degradation by plasma arginase. We hypothesized that serum LDH may represent a convenient biomarker of intravascular hemolysis and NO bioavailability, characterizing a clinical subphenotype of hemolysis-associated vasculopathy. In a cohort of 213 patients with sickle cell disease, we found statistically significant associations of steady-state LDH with low levels of hemoglobin and haptoglobin and high levels of reticulocytes, bilirubin, plasma hemoglobin, aspartate aminotransferase, arginase, and soluble adhesion molecules. LDH isoenzyme fractionation confirmed predominance of LD1 and LD2, the principal isoforms within erythrocytes. In a subgroup, LDH levels closely correlated with plasma cell-free hemoglobin, accelerated NO consumption by plasma, and impaired vasodilatory responses to an NO donor. Remarkably, this simple biomarker was associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in patients with sickle cell disease. We propose that LDH elevation identifies patients with a syndrome of hemolysis-associated NO resistance, endothelial dysfunction, and end-organ vasculopathy.
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PMID:Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. 1629 95

Gas chromatographic-mass spectral analysis of extracts obtained from in vitro culture of isolated retrocerebral complexes obtained from adult females of the moth Heliothis virescens resulted in identification of methyl farnesoate as well as juvenile hormone III (JH III) but not JH III acid. Inhibition of JH biosynthesis by incubation of tissue in synthetic Manduca sexta allatostatin (Manse-AST, pGlu-Val-Arg-Phe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-COOH) reduced production of these chemicals to negligible levels. However, incubation of tissue in the presence of Manse-AST plus farnesol resulted in production of significant amounts of both methyl farnesoate and JH III. Tissue incubated in the presence of Manse-AST plus methyl farnesoate produced only JH III. The results indicated that methyl farnesoate is naturally produced by the corpora allata of adult females of Heliothis virescens. However, tissue incubated in the presence of Manse-AST plus JH III acid also produced JH III in amounts equivalent to that produced by tissue incubated with methyl farnesoate. Thus, both methyl farnesoate and JH III acid could serve as a precursor for biosynthesis of JH III.
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PMID:Identification of methyl farnesoate from in vitro culture of the retrocerebral complex of adult females of the moth, Heliothis virescens (Lepidoptera: Noctuidae) and its conversion to juvenile hormone III. 1641 51

We report a 3-year-old Italian patient with the hyperornithinaemia, hyperammonaemia, homocitrullinuria (HHH) syndrome who presented with neurological deterioration after an intercurrent infection. Hyperammonaemia, coagulopathy and moderate hypertransaminasaemia were detected on hospital admission. Severe hepatocellular necrosis with hypertransaminasaemia (aspartate aminotransferase 20,000 UI/L, alanine aminotransferase 18,400 UI/L) and coagulopathy (PT < 5%) rapidly developed within few days, prompting evaluation for liver transplantation. A protein-restricted diet and arginine supplementation were immediately started, with a rapid improvement of the patient's neurological conditions and normalization of liver function tests and blood ammonia. The diagnosis of HHH syndrome was based on the presence of the typical metabolic abnormalities. Molecular analysis of the SLC25A15 gene showed that the patient was heterozygous for two novel mutations (G113C and M273K). The diagnosis of HHH syndrome should be considered in patients with fulminant hepatitis-like presentations. Early identification and treatment of these patients can be life-saving and can avoid liver transplantation.
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PMID:HHH syndrome (hyperornithinaemia, hyperammonaemia, homocitrullinuria), with fulminant hepatitis-like presentation. 1660 89

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a kind of inborn errors of metabolism, with the main clinic manifestations of jaundice, hepatomegaly, and abnormal liver function indices. As a mitochondrial solute carrier protein, citrin plays important roles in aerobic glycolysis, gluconeogenesis, urea cycle, and protein and nucleotide syntheses. Therefore citrin deficiency causes various and complicated metabolic disturbances, such as hypoglycemia, hyperlactic acidemia, hyperammonemia, hypoproteinemia, hyperlipidemia, and galactosemia. This paper reported a case of NICCD confirmed by mutation analysis of SLC25A13, the gene encoding citrin. The baby (male, 6 months old) was referred to the First Affiliated Hospital with the complaint of jaundice of the skin and sclera, which it had suffered from for nearly 6 months. Physical examination showed obvious jaundice and a palpable liver 5 cm below the right subcostal margin. Liver function tests revealed elevated enzymatic activities, like GGT, ALP, AST, and ALT, together with increased levels of TBA, bilirubin (especially conjugated bilirubin), and decreased levels of total protein/albumin and fibrinogen. Blood levels of ammonia, lactate, cholesterol, and triglyceride were also increased, and in particular, the serum AFP level reached 319,225.70 microg/L, a extremely elevated value that has rarely been found in practice before. Tandem mass analysis of a dried blood sample revealed increased levels of free fatty acids and tyrosine, methionine, citrulline, and threonine as well. UP-GC-MS analysis of the urine sample showed elevated galactose and galactitol. The baby was thus diagnosed with suspected NICCD based on the findings. It was then treated with oral arginine and multiple vitamins (including fat-soluble vitamins A, D, E, and K), and was fed with lactose-free and medium-chain fatty acids enriched formula instead of breast feeding. After half a month of treatment, the jaundice disappeared, and the laboratory findings, including liver function indices, blood levels of ammonia, lactate and AFP, were returned to normal level. The baby was followed up for 6 months. It developed well, and the abnormal laboratory findings, including MS-MS and UP-GC-MS analysis results, have been corrected, except a slightly elevated lactate level sometimes. SLC25A13 gene mutation analysis for the patient revealed a compound heterozygote of mutation 851del4 and 1638ins23 and therefore NICCD was definitely diagnosed.
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PMID:[A difficult and complicated case study: neonatal intrahepatic cholestasis caused by citrin deficiency]. 1661 6


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