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)

Two peptide inhibitors of juvenile hormone biosynthesis, designated G. bimaculatus allatostatins A1 and A2, have been purified from extracts of the brain of the field cricket Gryllus bimaculatus. The primary structures of these peptides were assigned as Ala-Gln-His-Gln-Tyr-Ser-Phe-Gly-Leu-NH2 (Grb-AST A1) and Ala-Gly-Gly-Arg-Gln-Tyr-Gly-Phe-Gly-Leu-NH2 (Grb-AST A2). Each of the peptides shows C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The two peptides are potent inhibitors of in vitro juvenile hormone production by corpora allata from virgin females of G. bimaculatus.
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PMID:Identification of two allatostatins from the cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae): additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis. 748 Aug 72

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with L-erythro-3-hydroxyaspartate (HOAsp) produces an intense absorption at 494 nm (epsilon = 13,650 M-1 cm-1), which is ascribed to the quinonoid intermediate. However, when Tyr70 of AspAT has been replaced by Phe, the enzyme shows only a faint absorption at 494 nm (epsilon = 522 M-1 cm-1) on the reaction with HOAsp. This indicates the involvement of the hydroxy group of Tyr70 in stabilizing the quinonoid intermediate formed from HOAsp and pyridoxal 5'-phosphate at the AspAT active site. Kinetic analysis of the absorption changes of the wild-type and Y70F mutant AspATs has shown that the reactions with HOAsp conform to the equation, EL + S<-->ES1<-->ES2<-->ES3<-->EM + P, in which there is a rapid formation of the quinonoid intermediate (ES2) from ES1, followed by a slow equilibrium between ES2 and ES3. ES3 absorbs primarily at 330 nm. The kinetic parameters for individual steps have been determined, and free energy profiles for the reactions of the two enzymes with HOAsp have been obtained. The stability of the quinonoid intermediates of the two enzymes in the normal catalytic reactions with aspartate has been assessed by static measurement of the spectra in the presence of both aspartate and oxalacetate, and the free energy profiles for the reactions have been similarly obtained. Comparison of the free energy levels in the profiles showed that the interaction of the beta-hydroxy group of HOAsp with the hydroxy group of Tyr70 accounts for 8.7 kJ mol-1 of the 18.5 kJ mol-1 stabilization of the quinonoid intermediate by the beta-hydroxy group. Model building of the active site of AspAT complexed with HOAsp suggests that the rest of the stabilization is mediated through the interaction of the beta-hydroxy group of HOAsp with the protonated epsilon-amino group of Lys258. This interaction is expected to strengthen the hydrogen-bonding network involving Tyr70, HOAsp, and the coenzyme phosphate. A similar network is possibly formed in the carbinolamine intermediate, suggesting ES3 to be the carbinolamine. A mechanism for the reaction of AspAT with HOAsp, which conforms to all the kinetic and spectroscopic data presented here, is proposed. This study provides a basis for subsequent spectroscopic characterization of the HOAsp-AspAT complex, which is a good model for the critical intermediate (quinonoid) structure of the AspAT-catalyzed reactions.
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PMID:Reaction of aspartate aminotransferase with L-erythro-3-hydroxyaspartate: involvement of Tyr70 in stabilization of the catalytic intermediates. 762 11

Four nonapeptides that inhibit juvenile hormone synthesis have been isolated by four high performance liquid chromatographic steps from extracts of the brain of the field cricket, Gryllus bimaculatus. The primary structures of these peptides were assigned by Edman degradation and mass spectrometry as Gly-Trp-Gln-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B1), Gly-Trp-Arg-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B2), Ala-Trp-Arg-Asp-Leu-Ser-Gly-Gly-Trp-NH2 (Grb-AST B3), and Ala-Trp-Glu-Arg-Phe-His-Gly-Ser-Trp-NH2 (Grb-AST B4). Each of the peptides shows high sequence similarity to the locustamyoinhibiting peptide (Lom-MIP), but is structurally different from all the allatostatins so far identified. The synthetic allatostatins Grb-AST B1-4 are potent inhibitors (50% inhibition at 10(-8) to 7 x 10(-8) M) of juvenile hormone III biosynthesis by corpora allata from 3-day-old virgin females of G. bimaculatus using an in vitro bioassay. At 10(-7) M, Grb-AST B1 also strongly inhibits juvenile hormone III biosynthesis by corpora allata from 2-day-old adult males and 1-day-old (males and females) and 4-day-old (females) last instar larvae of G. bimaculatus. The inhibitory effect of Grb-AST B1 was also evident on corpora allata from a related species, Acheta domesticus. Inhibition of juvenile hormone synthesis by Grb-AST B1-4 is reversible.
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PMID:A family of neuropeptides that inhibit juvenile hormone biosynthesis in the cricket, Gryllus bimaculatus. 767 41

Fulicin (Phe-D-Asn-Glu-Phe-Val-NH2) is an endogenous neuropeptide containing a D-amino acid from ganglia of the African giant snail Achatina fulica. We have cloned a novel cDNA (1,995 nucleotides) encoding a fulicin precursor from the snail cerebral and subesophageal ganglia. The fulicin precursor protein (357 amino acids) contains one copy of fulicin and at least nine other putative alpha-amidated neuropeptides composed of four to six amino acid residues. Seven of the nine neuropeptides were novel, and the other two had the same structures as Mytilus inhibitory peptide-related peptides previously isolated from the ganglia of Helix pomatia. All sequences of 10 peptides are flanked by Lys-Arg(Lys) at the N-terminus and by Gly-Lys-Arg(Lys) at the C-terminus. Nucleotide sequence analysis revealed that D-Asn present in fulicin is encoded by the usual L-Asn codon (AAT). Although fulicin has as yet only been isolated from the central ganglia. RNA blot analysis revealed that single transcripts of approximately 2.0 kb in size also exist in the ventricles and atria. These results suggest that fulicin and related peptides are produced in neurons and the heart by the processing of a ribosomally made precursor, although the mechanism of in-chain epimerization remains unclear.
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PMID:A novel cDNA sequence encoding the precursor of the D-amino acid-containing neuropeptide fulicin and multiple alpha-amidated neuropeptides from Achatina fulica. 772 9

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.
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PMID:Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms. 780 54

The production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.
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PMID:Structure-activity studies of allatostatin 4 on the inhibition of juvenile hormone biosynthesis by corpora allata: the importance of individual side chains and stereochemistry. 785 67

hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.
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PMID:Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses. 788 15

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.
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PMID:NMR studies of 1H resonances in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli. 796 37

Serum amino acid (AA) profiles are altered in epilepsy. It is not clear whether this is due to the disease process itself or to other variables such as seizure type, seizure frequency, duration of illness, medication, or altered liver function. We investigated serum AA profiles and liver enzymes in 73 epileptic patients and 90 healthy subjects and evaluated the data by analysis of variance to discriminate between age, sex, seizure type, duration of illness, seizure frequency, antiepileptic drug (AED) and increased serum liver enzyme levels, and their putative interaction with the serum AA profile. There was no correlation between the changes in the AA profile and age, duration of illness, seizure frequency, and seizure type. Seventy-two percent of the AED-treated patients and 33% of the unmedicated patients showed an increase in one or several serum liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and/or gamma-glutamyl transferase (gamma-GT)]; particularly gamma-GT. We observed a significant increase in serum concentrations of glutamine and glycine and decreased levels of taurine, threonine, serine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, tryptophan, and arginine in AED-treated patients but not in unmedicated patients. These results show that the changes in the serum AA profiles of epileptic patients treated with AEDs occur in patients with alteration of serum liver enzymes; whether this implies a causal relation is still uncertain.
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PMID:Serum amino acids, liver status, and antiepileptic drug therapy in epilepsy. 809 92

The Schiff base formed between Lys258 of Escherichia coli aromatic amino acid aminotransferase (ArAT) and the coenzyme pyridoxal 5'-phosphate (PLP) has a pKa value of 6.65. The pH dependency of the kinetic parameters was consistent with a mechanism by which the enzymatic form with the nonprotonated Schiff base productively binds aspartate, phenylalanine, and tryptophan. The Schiff base pKa value rose by 1.7-2.1 unit on binding of substrate analogs, and this strongly suggested protonation of the Schiff base upon formation of the Michaelis complex with substrates. The protonated "internal" Schiff base in the Michaelis complex is supposed to be attacked by the deprotonated substrate amino group, and this explains excellently the mechanism of transaldimination to form the PLP-substrate Schiff base. Phenylpropionate and indolepropionate caused similar increases in the pKa value to maleate. [Arg292-->Ala] ArAT showed the same pKa value as the wild-type enzyme. Therefore, neutralization of Arg292 by omega-carboxylate of dicarboxylic ligands, which had been well documented in aspartate aminotransferase to increase the Schiff base pKa, has little effect on the protonation of the Schiff base in ArAT. Thus the structure of ArAT is deliberately organized so that the Schiff base pKa is effectively modulated by substrates having only one carboxylate group.
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PMID:Protonation state of the active-site Schiff base of aromatic amino acid aminotransferase: modulation by binding of ligands and implications for its role in catalysis. 818 25


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