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Jorge E. Churchich - One of the best experts on this subject based on the ideXlab platform.
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Gabaculine and m-carboxyphenyl-pyridoxamine 5-phosphate as probes of the catalytic binding sites of 4-Aminobutyrate Aminotransferase.
European journal of biochemistry, 2005Co-Authors: Doo Sik Kim, Udoudo Moses, Jorge E. ChurchichAbstract:A homogeneous 4-Aminobutyrate Aminotransferase isolated from pig brain exhibits a kcat of 9.6 s−1 and contains one mole of pyridoxal 5-phosphate/mole of dimer. The reaction of the enzyme with gabaculine (5-amino-1, 3-cyclohexadiene carboxylic acid) was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5-phosphate. Stopped-flow kinetic studies reveal that gabaculine reacts with the cofactor bound to the Aminotransferase with a second-order rate constant of 2.5 × 103 M−1s−1. Fluorometric titrations of the apoprotein with m-carboxyphenyl-pyridoxamine 5-phosphate show the binding of two moles of inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10-fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dimer, but the binding site characterized by a weaker affinity constant for pyridoxal 5-phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.
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Recombinant brain 4-Aminobutyrate Aminotransferases overexpression, purification, and identification of Lys-330 at the active site
Biochimica et biophysica acta, 1997Co-Authors: Young Tae Kim, Young Hwan Song, Jorge E. ChurchichAbstract:4-Aminobutyrate Aminotransferase (4-Aminobutyrate: 2-oxoglutarate Aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4-Aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4-Aminobutyrate Aminotransferase, we analyzed 4-Aminobutyrate Aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-Aminobutyrate Aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P. To verify the catalytic site of 4-Aminobutyrate Aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4-Aminobutyrate Aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-Aminobutyrate Aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4-Aminobutyrate Aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the Aminotransferase.
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SCREENING AND SEQUENCE DETERMINATION OF A CDNA ENCODING THE HUMAN BRAIN 4-Aminobutyrate Aminotransferase
Gene, 1995Co-Authors: Yaa Difie Osei, Jorge E. ChurchichAbstract:Abstract A human brain cDNA library constructed in the λZAP®II vector was screened using a fragment of pig brain cDNA encoding 4-Aminobutyrate Aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.
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Characterization of monomeric 4-Aminobutyrate Aminotransferase at low pH.
European journal of biochemistry, 1995Co-Authors: Teresa Pineda, Yaa Difie Osei, Jorge E. ChurchichAbstract:4-Aminobutyrate Aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, фobs= 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL≈ 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-Aminobutyrate Aminotransferase in the cell.
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Unfolding of 4-Aminobutyrate Aminotransferase equilibrium and kinetic studies
Biochimica et biophysica acta, 1994Co-Authors: Teresa Pined, Jorge E. ChurchichAbstract:Abstract The unfolding of pig liver 4-Aminobutyrate Aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-Aminobutyrate Aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the Aminotransferase unfolds in a single kinetic phase. Unfolded 4-Aminobutyrate Aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two- state model for the unfolding of the Aminotransferase is proposed.
Anne M Evans - One of the best experts on this subject based on the ideXlab platform.
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2 pyrrolidinone and succinimide as clinical screening biomarkers for gaba transaminase deficiency anti seizure medications impact accurate diagnosis
Frontiers in Neuroscience, 2019Co-Authors: Adam D Kennedy, William E Craigen, Kirk L Pappan, Taraka R Donti, Reid V Sutton, Mauricio R Delgado, Seema R Lalani, Toni S Pearson, Marwan Shinawi, Anne M EvansAbstract:: Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.
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Data_Sheet_1_2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis.xlsx
2019Co-Authors: Adam D Kennedy, William E Craigen, Kirk L Pappan, Taraka R Donti, Reid V Sutton, Mauricio R Delgado, Seema R Lalani, Toni S Pearson, Marwan Shinawi, Anne M EvansAbstract:Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.
Cornelis Jakobs - One of the best experts on this subject based on the ideXlab platform.
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A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy
Journal of Inherited Metabolic Disease, 2010Co-Authors: Megumi Tsuji, Noriko Aida, Takayuki Obata, Moyoko Tomiyasu, Noritaka Furuya, Kenji Kurosawa, Abdellatif Errami, K. Michael Gibson, Gajja S. Salomons, Cornelis JakobsAbstract:Background Deficiency of 4-Aminobutyrate Aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy (^1H-MRS). Methods Neuroimaging was performed at the first episode of lethargy. For ^1H-MRS, locations were placed in the semioval center and the basal ganglia. Quantification of metabolite concentrations were derived using the LCModel. We confirmed the diagnosis subsequently by enzyme and molecular studies, which involved direct DNA sequence analysis and the development of a novel multiplex ligation-dependent probe amplification test. Results ^1H-MRS analysis revealed an elevated GABA concentration in the basal ganglia (2.9 mmol/l). Based on the results of quantitative ^1H-MRS and clinical findings, GABA-T deficiency was suspected and confirmed in cultured lymphoblasts. Molecular studies of the GABA-T gene revealed compound heterozygosity for a deletion of one exon and a missense mutation, 275G>A, which was not detected in 210 control chromosomes. Conclusions Our results suggest that excessive prenatal GABA exposure in the central nervous system (CNS) was responsible for the clinical manifestations of GABA transaminase deficiency. Our findings suggest the dual nature of GABA as an excitatory molecule early in life, followed by a functional switch to an inhibitory species later in development. Furthermore, quantitative ^1H-MRS appears to be a useful, noninvasive tool for detecting inborn errors of GABA metabolism in the CNS.
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A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy
Journal of inherited metabolic disease, 2010Co-Authors: Megumi Tsuji, Noriko Aida, Takayuki Obata, Moyoko Tomiyasu, Noritaka Furuya, Kenji Kurosawa, Abdellatif Errami, K. Michael Gibson, Gajja S. Salomons, Cornelis JakobsAbstract:Background Deficiency of 4-Aminobutyrate Aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy (1H-MRS).
Adam D Kennedy - One of the best experts on this subject based on the ideXlab platform.
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2 pyrrolidinone and succinimide as clinical screening biomarkers for gaba transaminase deficiency anti seizure medications impact accurate diagnosis
Frontiers in Neuroscience, 2019Co-Authors: Adam D Kennedy, William E Craigen, Kirk L Pappan, Taraka R Donti, Reid V Sutton, Mauricio R Delgado, Seema R Lalani, Toni S Pearson, Marwan Shinawi, Anne M EvansAbstract:: Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.
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Data_Sheet_1_2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis.xlsx
2019Co-Authors: Adam D Kennedy, William E Craigen, Kirk L Pappan, Taraka R Donti, Reid V Sutton, Mauricio R Delgado, Seema R Lalani, Toni S Pearson, Marwan Shinawi, Anne M EvansAbstract:Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.
Robert A. John - One of the best experts on this subject based on the ideXlab platform.
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Determinants of Substrate Specificity in ω-Aminotransferases
The Journal of biological chemistry, 2005Co-Authors: Michaela Markova, Caroline Peneff, Miichael J. E. Hewlins, Tilman Schirmer, Robert A. JohnAbstract:Ornithine Aminotransferase and 4-Aminobutyrate Aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine Aminotransferase and (ii) that, uniquely among related, structurally characterized Aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine Aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in Aminobutyrate Aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-Aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.
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Primary Structure and Tissue Distribution of Human 4‐Aminobutyrate Aminotransferase
European journal of biochemistry, 1995Co-Authors: Daniela De Biase, Donatella Barra, Maurizio Simmaco, Robert A. John, Francesco BossaAbstract:cDNA encoding human 4-Aminobutyrate Aminotransferase (Aminobutyrate:2-oxoglutarate Aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.-Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267-1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-Aminobutyrate Aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.
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Protein structure of pig liver 4‐Aminobutyrate Aminotransferase and comparison with a cDNA‐deduced sequence
European journal of biochemistry, 1992Co-Authors: Daniela De Biase, Donatella Barra, Francesco Bossa, Bruno Maras, Robert A. JohnAbstract:The amino acid sequence of pig liver 4-Aminobutyrate Aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215–7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107–142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.
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protein structure of pig liver 4 Aminobutyrate Aminotransferase and comparison with a cdna deduced sequence
FEBS Journal, 1992Co-Authors: Daniela De Biase, Donatella Barra, Francesco Bossa, Bruno Maras, Robert A. JohnAbstract:The amino acid sequence of pig liver 4-Aminobutyrate Aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215–7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107–142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.
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Chemistry of the inactivation of 4-Aminobutyrate Aminotransferase by the antiepileptic drug vigabatrin.
The Journal of biological chemistry, 1991Co-Authors: Daniela De Biase, Francesco Bossa, D. Barra, Piero Pucci, Robert A. JohnAbstract:Abstract The chemical modification of pig liver 4-Aminobutyrate Aminotransferase by the antiepileptic drug 4-aminohex-5-enoate (Vigabatrin) has been studied. After inactivation by 14C-labeled Vigabatrin, the enzyme was digested with trypsin, and automated Edman degradation of the purified labeled peptide gave the sequence FWAHEHWGLDDPADVMTFSKK. Chymotryptic digestion of the tryptic peptide and sequencing of a resulting tripeptide identified the penultimate lysine residue of this peptide as the site of covalent modification. This lysine normally binds the coenzyme. Absorption spectroscopy demonstrated the absence of coenzyme from the tryptic peptide, and mass spectrometry showed its mass/charge ratio to be increased by 128. All of the bound coenzyme released after denaturation of the inactivated enzyme was as pyridoxamine phosphate. The structural nature of the modification is deduced, and mechanisms for its occurrence identified. Initially, 1 mol of radiolabeled inhibitor was bound per mol of monomer of the enzyme, although approximately half was released during denaturation and digestion, while the remainder was irreversibly bound. Coenzyme not released as pyridoxamine phosphate retained the absorbance characteristics of the aldimine, although the enzyme was completely inactive. Mass spectrometry of the sample of purified radiolabeled tryptic peptide revealed the presence of an approximately equal amount of a second fragment that contained no modification and from which the second lysine was absent, indicating that at the time of proteolysis the active site lysine was unaltered in 50% of the enzyme molecules.
