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At least 109 records · Page 6Linked to original sources
PubMed1985

Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane.

The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.

Aspartic Acid↗
PubMed1992

Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis.

We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.

Amino Acid Sequence↗
PubMed1993

Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity.

Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.

Amino Acid Sequence↗
PubMed1993

Lysine 319 interacts with both glutamic acid 269 and aspartic acid 240 in the lactose carrier of Escherichia coli.

It is believed that there are several charged amino acid residues in membrane-spanning alpha-helices of the lactose carrier of Escherichia coli. Evidence has previously been presented for two different salt bridges in membrane-spanning regions of the lactose carrier. One of these involves an interaction between Asp-237 and Lys-358; another involves interaction between Asp-240 and Lys-319. Additional studies of Lys-319 suggest that it may interact with Glu-269 as well as Asp-240. A cell containing the LacY gene with the mutation Lys-319-->Asn failed to ferment melibiose and after several days melibiose-positive mutants arose on indicator plates. These revertants showed second site mutations which replaced Asp-240 by neutral amino acids (Val or Gly). In addition, a second site mutation showed Glu-269 changed to Asn. Cells containing the mutation Lys-319-->Leu also failed to ferment melibiose and melibiose-positive revertants showed Asp-240-->Ala and Asp-240-->Tyr as well as Tyr-236-->Phe and His-322-->Arg. Second site revertants were also sought from the mutant Glu-269-->Asn which grew poorly on melibiose minimal plates. Melibiose-positive revertants included the double mutant Gln-269/Asn-319. All of the Glu-269-->Asn mutants were extremely defective in transport. It was concluded that Lys-319 interacts with Glu-269 and Asp-240 probably as salt bridges.

Amino Acid Sequence↗
PubMed1987

The inhibition by morphine and D-aspartic acid of antibody production against Salmonella typhimurium antigen in rats: its antagonism by L-aspartic acid.

The changes in the production of antibody against Salmonella typhimurium antigen were investigated in rats by means of the agglutination test after chronic oral administration of the L-asparaginase inhibitors morphine (M) or D-aspartic acid (D-Asp) alone or together with L-aspartic acid (L-Asp) and food restriction, all of which had been started five days before the injections of antigen. The statistical evaluation, carried out after the titers had been defined as -log2 of the highest dilution giving a positive agglutination reaction, showed that M or D-Asp significantly decreased antibody production in comparison with the immunized control or food restricted group. The concomitant administration of L-Asp appeared to significantly antagonize the inhibitory effect of both M and D-Asp. Therefore, the results were considered as further supporting evidence for the fact that the deleterious effect of M on the immune system and its functions might be related to the inhibitory effect of M on L-asparaginase activity.

Administration, Oral↗
PubMed2000

An in situ Infrared Spectroscopic Study of Glutamic Acid and of Aspartic Acid Adsorbed on TiO(2): Implications for the Biocompatibility of Titanium.

In situ ATR-IR spectroscopy has been applied to the study of glutamic (Glu) and aspartic (Asp) acid adsorbed on amorphous TiO(2) particle films. Unlike Asp, which gives evidence of one major adsorbed species, Glu yields several spectroscopically distinct structures upon adsorption to TiO(2). The pH dependence of Glu and Asp adsorption is also different, with Glu adsorbing markedly to TiO(2) at pH where electrostatic interactions between the surface and adsorbate are unfavorable. Application of the Langmuir model to adsorption isotherms yields a single binding constant for Asp and two binding constants for Glu, further supporting the evidence for different adsorbed Glu species. This is the first investigation of the molecular structure of Glu and Asp species adsorbed on amorphous TiO(2) using in situ ATR-IR spectroscopy. Copyright 2000 Academic Press.

Journal Article↗
PubMed1989

AMPA, kainic acid, and N-methyl-D-aspartic acid stimulate locomotor activity after injection into the substantia innominata/lateral preoptic area.

The substantia innominata/lateral preoptic area (SI/LPO) is a subpallidal region which has been shown to regulate the hypermotility produced by drugs acting in the nucleus accumbens. Evidence has been presented that the SI/LPO contains glutamatergic nerve terminals and receptors for excitatory amino acids. The purpose of this study was to determine the effects of the activation of excitatory amino acid receptors in the SI/LPO on locomotor activity following the direct injection of excitatory amino acids into this brain site. It was found that the bilateral injection of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainic acid, and N-methyl-D-aspartic acid into the SI/LPO produced marked dose-dependent stimulations of locomotor activity which resembled the effects of these agents after their injection into the nucleus accumbens. The effect, however, was bell-shaped in that at high doses, the locomotor activity values decreased from their peak values. The coinjection of gamma-glutamylaminomethylsulfonate (GAMS) with AMPA into the SI/LPO was found to inhibit the hypermotility response to AMPA at doses that were unable to produce a significant inhibition of the hypermotility responses to kainic acid or N-methyl-D-aspartic acid. The injection of 6,7-dinitroquinoxaline-2,3-dione (DNQX) into the SI/LPO inhibited the hypermotility responses to AMPA or kainic acid while having no significant inhibitory effect on N-methyl-D-aspartic acid stimulated locomotor activity. The injection of D-alpha-aminoadipic acid into the SI/LPO produced a significant inhibition of the hypermotility response produced by N-methyl-D-aspartic acid at a dose that did not produce a significant inhibition of the hypermotility response produced by AMPA.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Aminoadipic Acid↗
PubMed1992

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.

Aspartic Acid↗
PubMed1989

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2.

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.

Animals↗
PubMed1982

Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus.

The effect of the cephalosporin precursors and amino acids of the aspartic acid family on antibiotic production by Streptomyces clavuligerus was investigated DL-meso-Diaminopimelate and L-lysine each stimulated specific antibiotic production by 75%. A fourfold increase in specific production was obtained by simultaneous addition of the two compounds. The stimulation could be further increased by adding valine to the two effectors. In the streptomycetes the alpha-aminoadipyl side chain of the cephalosporin antibiotics is derived from lysine. Streptomycetes, like other bacteria, are expected to produce lysine from aspartic acid; therefore, the feedback control mechanisms operating in the aspartic acid family pathway of S. clavuligerus, which may affect the flow of carbon to alpha-aminoadipic acid, were investigated. Threonine inhibited antibiotic production by 41% when added to minimal medium at a concentration of 10 mM. Simultaneous addition of 10 mM lysine completely reversed this inhibition. The aspartokinase of S. clavuligerus was found to be subject to concerted feedback inhibition by threonine and lysine. Threonine may act to limit the supply of lysine available for cephamycin C biosynthesis via this concerted mechanism. Single or simultaneous addition of any other amino acid of the aspartate family in the in vitro assay did not inhibit aspartokinase activity. Activity was stimulated by lysine. Aspartokinase biosynthesis was partially repressed by methionine or isoleucine at concentrations higher than 10 mM. Methionine, but not isoleucine, inhibited cephamycin C synthesis by 27% when added to minimal medium at a concentration of 10 mM. Dihydrodipicolinate synthetase, the first specific enzyme of the lysine branch, was not inhibited by lysine but was partially inhibited by high concentrations of 2,6-diaminopimelate and alpha-aminoadipate; it was slightly repressed by diaminopimelic acid. Homoserine dehydrogenase activity was inhibited by threonine and partially repressed by isoleucine. It appears that S. clavuligerus aspartokinase is a key step in the control of carbon flow toward alpha-aminoadipic acid.

2-Aminoadipic Acid↗
PubMed1987

Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.

ADP Ribose Transferases↗