Electron-microscopic investigation of Escherichia coli glutamate decarboxylase

Tikhonenko, Anna S.; Sukhareva, B. S.; Braunstein, A. E.

doi:10.1016/0005-2744(68)90232-5

Cited by 12 publications

(7 citation statements)

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“…Gale (14) proposed a general role for the inducible bacterial amino acid decarboxylases, including GAD, in the maintenance of physiological pH under acidic conditions. E. coli GAD has been extensively characterized with respect to its biophysical and biochemical properties (1,14,30,31,(39)(40)(41)(42)(43)(44)(45)(46), and a partial protein sequence has been available for some time (42,43). Based on genetic linkage studies in E. coli, the structural gene for GAD (gadS) and a potential regulatory gene (gadR) have been mapped between mtl at approximately 80.7 minutes and gltS at approximately 82.4 minutes (3,23,26,27), but these early results have not been followed up.…”

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Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci

et al. 1992

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Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD a and GAD 13, and these differed by only five amino acids. Both GAD a and GAD 13 contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gad4 and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.The enzyme glutamate decarboxylase (GAD; also known as glutamic acid decarboxylase; EC 4.1.1.15) catalyzes the a-decaboxylation of glutamic acid to produce -y-aminobutyric acid. Within bacteria, GAD activity seems to be relatively unique to Escherichia coli (37). Gale (14) proposed a general role for the inducible bacterial amino acid decarboxylases, including GAD, in the maintenance of physiological pH under acidic conditions. E. coli GAD has been extensively characterized with respect to its biophysical and biochemical properties (1,14,30,31,(39)(40)(41)(42)(43)(44)(45)(46), and a partial protein sequence has been available for some time (42, 43). Based on genetic linkage studies in E. coli, the structural gene for GAD (gadS) and a potential regulatory gene (gadR) have been mapped between mtl at approximately 80.7 minutes and gltS at approximately 82.4 minutes (3,23,26,27), but these early results have not been followed up. For the purposes of our immunological studies, we were interested in obtaining the complete DNA and protein sequences of E. coli GAD. At the time this work was initiated, an extensive search of the literature and all available sequence data bases suggested that neither the complete protein sequence nor the gene sequence for E. coli GAD had been determined. To obtain this information, we generated a DNA probe for the gene based on a recent more extensive partial protein sequence (24). By using this probe on E. coli genomic DNA, we discovered that there were not one but two separate cross-hybridizing GAD genes. We report here the complete DNA sequences and map pos...

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Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci

et al. 1992

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“…For the best studied system, Escherichia coli glutamate decarboxylase, only the first part of the physiological reaction, up to carbon dioxide desorption, has been examined (O'Leary et al, 1981;Abell & O'Leary, 1988a). The enzyme shows rather broad titration curves for V and V/K (O'Leary et al, 1970;Fonda, 1972), and further studies are hampered because the active hexameric form of the protein dissociates into dimers above pH 6.0 (Tikhonenko et al, 1968;Sukhareva, 1986).…”

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Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination

Akhtar

Stevenson²,

Gani³

1990

Biochemistry

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L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…The latter authors proposed that the enzyme was a dimer of mol wt 300,000 whose properties could be profoundly affected by protein concentration and temperature. Recent electron microscopic investigation (Tikhonenko et al, 1968) has indicated that the enzyme has a hexameric structure that can be disrupted by dilution at low temperature.…”

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