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At least 163 records · Page 9Linked to original sources
PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗
PubMed2000

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.

Amino Acid Sequence↗
PubMed1986

Blockade of N-methyl-D-aspartate-sensitive acidic amino acid receptors inhibits ischemia-induced accumulation of purine catabolites in the rat striatum.

The effect of blocking N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid (EAA) receptors during brain ischemia was studied in order to test a link between EAAs and neuronal energy metabolism. The receptors were blocked unilaterally in the rat striatum before, during and after an ischemic insult. The receptor blocker, D-2-amino-5-phosphonovalerate (D-APV) was administered by dialysis perfusion, which also allowed continuous sampling for analysis of adenosine triphosphate degradation products, i.e. purine catabolites, in control and D-APV-treated striata. Purine catabolites were analysed with reversed-phase liquid chromatography. Hypoxanthine, xanthine, inosine and adenosine increased dramatically in the striatum during ischemia and reached maximum levels during early reperfusion. D-APV reduced the extracellular accumulation of all measured purine catabolites during ischemia/reflow and improved to some extent the recovery of the striatal electroencephalographic activity in the majority of the animals. The results suggest that NMDA receptor blockade attenuates acute changes in energy metabolism during ischemia.

2-Amino-5-phosphonovalerate↗
PubMed1995

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.

Aspartic Acid↗
PubMed1995

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: effects of acidic amino acid substitutions.

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40-->Asp substitution.

3T3 Cells↗
PubMed1998

Analysis of organic acids, amino acids, and carnitine in dogs with lipid storage myopathy.

Abnormal accumulations of lipid droplets, localized predominantly in histochemical type 1 fibers, were observed in fresh frozen sections of muscle biopsies from 25 dogs with myalgia, weakness, and muscle atrophy. Compared to controls, lactic acidemia, hyperalaninemia, lactic and pyruvic aciduria, variably increased urinary excretion of carnitine esters, and muscle carnitine deficiency were present. These findings support a metabolic block in oxidative metabolism resulting in lactic acidemia in dogs with lipid storage myopathy.

Acids↗
PubMed2002

Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport.

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC ) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif ((213)YTGGTTGVAKGA(224) and (356)GYGLTE(361)) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD(G216A), fadD(T217A), fadD(G219A), and fadD(K222A)) and 2) those with 10% or less wild-type activity (fadD(Y213A), fadD(T214A), and fadD(E361A)). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

Adenosine Monophosphate↗