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)

Aromatic amino acid aminotransferase (ArAT) from Escherichia coli was overexpressed in E. coli cells, purified, and characterized. The enzyme was similar to aspartate aminotransferase (AspAT) of E. coli in many aspects, such as gross protein structure and spectroscopic properties. The reactions of pyridoxal 5'-phosphate-form ArAT with amino acids and pyridoxamine 5'-phosphate-form ArAT with oxo acids were investigated using stopped-flow spectrophotometric techniques. The kinetic parameters for these "half" reactions could excellently explain the ArAT-catalyzed overall transamination reactions at pH 8.0. Reactions of ArAT with aspartate and tryptophan which had been deuterated at position 2 showed isotope effects of 2.5 and 6.0 in the kcat values of the half-reactions, showing that the proton-transfer step is at least partially rate-limiting for these reactions. ArAT and AspAT showed overlapping substrate specificity. Both ArAT and AspAT were active toward dicarboxylic substrates. ArAT showed, however, 10(3)-fold higher activity toward aromatic substrates than AspAT. This high activity toward aromatic substrates was in part ascribed to the active site hydrophobicity of ArAT, which was suggested to be about 1.4 times as large as that of AspAT. In addition to dicarboxylic substrate analogs, aromatic substrate analogs such as carboxylic acids, 2-methyl amino acids, and 3-hydroxy amino acids caused characteristic changes in the absorption spectra of ArAT, while these aromatic analogs did not significantly change the spectra of AspAT. In particular, the erythro-3-hydroxy analogs of phenylalanine and aspartate caused a prominent absorption of ArAT at around 500 nm, which is generally ascribed to the accumulation of quinonoid intermediates. The threo forms of these 3-hydroxy analogs acted as substrates for ArAT. The erythro and threo forms of 3-hydroxyaspartate reacted with AspAT similarly as they reacted with ArAT; however, both forms of 3-phenylserine were poor substrates for AspAT, although phenylalanine was a fairly good substrate for AspAT. The observations on the two erythro-3-hydroxy amino acids show the similar orientation of these analogs in the active site of ArAT, probably through a hydrogen-bonding network involving the hydroxy groups of the analogs and Tyr70, and suggest that the aromatic binding pocket is near or even overlaps the side-chain-carboxylate-binding site for dicarboxylic substrates.
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PMID:Escherichia coli aromatic amino acid aminotransferase: characterization and comparison with aspartate aminotransferase. 821

The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy. 834 9

Neutrophil function was studied in rats with common bile duct ligation. Superoxide production stimulated by phorbol myristate acetate, opsonized zymosan or formyl-methionyl-leucyl-phenylalanine; phagocytosis; and chemotaxis were significantly greater in neutrophils from rats with common bile duct ligation than in sham-operated control rats. Enhanced neutrophil activity was observed within 12 hr of bile duct ligation; it remained increased during the 15-day study. Preincubation of neutrophils from control rats with sera of rats with common bile duct ligation did not increase superoxide generation. This suggests that the high superoxide production observed in neutrophils of rats with common bile duct ligation was not an immediate effect of the serum. Neutrophils of rats with portal vein ligation exhibited normal activity, indicating that portal systemic shunting per se is not the underlying mechanism for increased activity. The elevated levels of AST and alkaline phosphatase, indicating liver damage, that appeared within 12 hr of bile duct ligation correlated with the increased superoxide generation.
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PMID:Increased neutrophil function induced by bile duct ligation in a rat model. 838 51

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase. 843 41

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis. 852 73

The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AAtase in which Tyr-225 is converted to Phe (Y225F) was investigated. The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm. The primary C alpha 2H-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AAtase at pH 8.9 and 7.5 on kcat/KMAsp are approximately 2, and the KIEs on kcat are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C alpha 2H-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values. The solvent deuterium KIEs (SKIEs) on kcat for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximately 2, and the SKIE on kcat/kMAsp is increased from 1.3 to 2.3 by the mutation. The C4' (S)-2H-pyridoxamine 5'-phosphate KIE values on reactions of alpha-ketoacids with both enzymes are near unity. The viscosity effects on kcat/KMAsp and kcat for wild-type AAtase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited. The viscosity effects on kcat/KMAsp and kcat for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited. A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AAtase. C alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F- catalyzed reaction.
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PMID:The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis. 861 15

Aberrations of the p53 and Rb tumour suppressor genes were examined in 12 human hepatocellular carcinoma (HCC)-derived cell lines from different geographic areas and 9 local HCCs by restriction fragment length polymorphisms (RFLP), polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCP) and DNA sequencing. The relationships between genetic changes and hepatitis B virus (HBV) DNA integration in samples were compared. None of the cell lines and tumours showed structural changes in the Rb gene, while 6 cell lines and 2 tumours had mutation or deletion in exons 5 to 8 of p53. Mutations include an AGG --> AGT (Arg --> Ser) transversion at codon 249 in PLC/PRF/5 and Mahlavu, an AAT --> AAA (Asn --> Cys) transversion at codon 200 in TONG/HCC, an AAG --> GAG (Lys --> Glu) transition at codon 139 in HCC-T, a CAT --> CGT (His --> Arg) transition at codon 214 in SC4, and a CCC --> CTC (Pro --> Leu) transition at codon 250 in SC8. In Huh4, an 18-bp deletion from codon 264 to 270 resulted in loss of Leu-Gly-Arg-Asn-Ser-Phe from the amino acid sequences 265 to 270, whereas Hep3B had a 7-kb deletion after exon 7 of p53. Our data indicate that whereas Rb may not have pleiotropic effects on HCC, p53 aberrations are frequently involved in hepatocarcinogenesis. Further, HBV infection appears to be unrelated to the micro-genetic changes of p53. The G to T codon-249-mutation is consistent with HCCs arising from areas at high risk for both aflatoxin B1 (AFB1) exposure and HBV infection.
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PMID:Tumour suppressor p53 and Rb genes in human hepatocellular carcinoma. 877 41

We found a gene homologous to tyrB, which encodes aromatic amino acid aminotransferase (ArAT, EC2.6.1.57) in Escherichia coli, in the genome of Salmonella typhimurium IFO 13245. The S. typhimurium tyrB product consists of 397 amino acid residues. The amino acid sequence shows 87.9% identity with that of E. coli ArAT, but shows lower identity (42.3%) with that of E. coli aspartate aminotransferase (AspAT, EC2.6.1.1). When the S. typhimurium tyrB gene was expressed in an E. coli mutant whose intrinsic tyrB gene had been inactivated, the activity of transaminating tyrosine and phenylalanine could be recovered, indicating that the S. typhimurium tyrB gene product possesses transamination activities similar to those of the E. coli ArAT. Elucidation of the molecular features of a new ArAT may be helpful for structural and functional analyses of ArAT and AspAT with regard to the different but overlapping substrate specificity of the two enzymes.
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PMID:Cloning and characterization of the tyrB gene from Salmonella typhimurium. 880 8

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of GpdhUF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the GpdhF (fast) allele. The GpdhUF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the XiamenUF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the IowaUF and NetherlandsUF alleles. The mobility of the RaleighUF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the BrazzavilleUF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the GpdhUF alleles originate from different mutational events, and only two of them--IowaUF and NetherlandsUF--might share a common ancestry. The GPDH activity of the IowaUF allele is intermediate between those of the GpdhS and GpdhF control stocks. The other GpdhUF variants have lower activities than the controls: XiamenUF--83%, RaleighUF--80% and BrazzavilleUF--73% of the GpdhF control.
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PMID:Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase 'ultra-fast' electrophoretic alleles in Drosophila melanogaster. 890 Nov 36

Eight myoinhibiting peptides were purified by high performance liquid chromatography from a methanolic extract of 7000 brains of the desert locust, Schistocerca gregaria. Complete sequences were obtained via a novel, combined approach employing: (1) chemical microsequencing and (2) post-source decay analysis on a reflectron time-of-flight mass spectrometer using matrix-assisted laser desorption/ionisation. Each of the peptides shows C-terminal amino acid sequence similarity to cockroach and cricket allatostatins and to blowfly callatostatins. Therefore, these novel peptides were designated Schistocerca gregaria allatostatins (Scg-ASTs) or schistostatins and their primary structures were determined to be: Ala-Tyr-Thr-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (Scg-AST-2), Ala-Thr-Gly-Ala-Ala-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-3), Gly-Pro-Arg-Thr-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-4), Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-5), Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-6), Ala-Gly-Pro-Ala-Pro-Ser-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-7), Glu-Gly-Arg-Met-Tyr-Ser-Phe-Gly-Leu-NH2 (Scg-AST-8), and Ala-Pro-Ala-Glu-His-Arg-Phe-Ser-Phe-Gly-Leu-NH2 (Scg-AST-10). Synthetic Scg-AST peptides inhibit the peristaltic movements of the oviduct of S. gregaria. Although all eight peptides show potent inhibitory effects on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diploptera punctata, no allatostatic effects were observed on CA of the desert locust (S. gregaria).
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PMID:Isolation and characterization of eight myoinhibiting peptides from the desert locust, Schistocerca gregaria: new members of the cockroach allatostatin family. 890 48


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