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PubMed1993

Functional roles of the invariant aspartic acid 55, tyrosine 306, and aspartic acid 309 in glucoamylase from Aspergillus awamori studied by mutagenesis.

Three mutants, Asp55-Gly, Tyr306-->Phe, and Asp309-->Asn, of Aspergillus awamori glucoamylase (identical to Aspergillus niger glucoamylase) were constructed to elucidate the roles of two conserved regions within fungal glucoamylases. Kinetic studies indicate that both of these regions are closely associated with activity. The Asp55-->Gly mutation decreases the kcat approximately 200 times toward maltose and isomaltose, while KM values remain similar to the wild-type. This localizes Asp55 to subsite 1 of glucoamylase where it affects catalytic activity, but not ground-state binding. The pKa value of the catalytic general acid, Glu179, is 1 pH unit lower in that mutant compared to wild-type enzyme, confirming the proximity of Asp55 to the site of catalysis. Tyr306-->Phe is highly active, but affects binding in subsite 2. It moreover shows enhanced binding in the fourth subsite, suggesting that the conserved region around residue 306 interacts with Trp120, a critical residue that directs conformational changes stabilizing the transition-state structure. Finally, the Asp309-->Asn mutation decreases the kcat for isomaltose hydrolysis around 200-fold, but only 30-fold for maltose. This specific effect on the hydrolysis of the alpha-1,6-linked substrate locates Asp309 to subsite 2. Substitution of Asp309 influences affinities of distant subsites, especially subsite 4, similar to mutations of other carboxylic acid residues situated near subsites 1 and 2.

Aspartic Acid↗
PubMed1983

Conformation and structure of acidic dipeptides. Crystal structures of L-alanyl-L-aspartic acid and alpha-L-glutamyl-L-aspartic acid.

The crystal structures of the dipeptides L-alanyl-L-aspartic acid, C7H12N2O5, and alpha-L-glutamyl-L-aspartic acid, C9H14N2O7, have been determined from three-dimensional X-ray diffractometer data. Alanylaspartic acid crystallizes in the orthorhombic space group P2(1)2(1)2(1) with four formula units in a cell of dimensions a = 13.389(5), b = 14.467(3), c = 4.781(1) A. Glutamylaspartic acid also crystallizes in space group P2(1)2(1)2(1) with four formula units in a cell of dimensions a = 13.709(5), b = 16.126(7), c = 4.939(5) A. Both structures were solved by direct methods and refined by full-matrix least squares methods; the final value of the weighted R-factors (on F) were 0.040 based on 790 independent intensities with I greater than or equal to 2 sigma (I) for Ala-Asp and 0.033 based on 1105 intensities with I greater than or equal to 2 sigma (I) for Glu-Asp. Each dipeptide occurs as a zwitterion with the amino terminus protonated and the main chain carboxyl group deprotonated. The conformation of the peptide linkage is trans in both molecules, the omega torsion angle being - 175.9 degrees in Ala-Asp and 174.3 degrees in Glu-Asp. There is considerable intermolecular, but not intramolecular, hydrogen bonding in the crystals. The conformations and structures of Ala-Asp and Glu-Asp are compared to those of other structurally characterized acidic dipeptides.

Dipeptides↗
PubMed1981

Amino acid esters of phenols as prodrugs: synthesis and stability of glycine, beta-aspartic acid, and alpha-aspartic acid esters of p-acetamidophenol.

pH-rate profiles were calculated for the hydrolysis of the glycine (II), beta-aspartic acid (III), and alpha-aspartic acid (IV) esters of p-acetamidophenol (I) at 25 degrees and mu = 1.0 M. The hydrolysis of esters II and III occurred predominantly via intermolecular reactions involving water, hydroxide ion, and the various ionic forms of the substrates. The hydrolysis of ester IV occurred predominantly via intramolecular reactions. The catalytic effects of formate, acetate, and phosphate ions as well as of tromethamine on the degradation on ester II were similar to their effects on the hydrolysis of ethyl dichloroacetate. The efficient catalysis of the hydrolysis of ester II in bicarbonate buffers was consistent with a mechanism that involved carbon dioxide and the formation and decomposition of a carbamate intermediate.

Acetaminophen↗
PubMed1994

Roles of aspartic acid 15 and 21 in glucagon action: receptor anchor and surrogates for aspartic acid 9.

The discovery of aspartic acid at position 9 in glucagon to be a critical residue for transduction has spurred renewed efforts to identify other strategic residues in the peptide sequence that dictate either receptor binding or biological activity. It also became apparent from further studies that Asp9 operates in conjunction with His1 in the activation mechanism that follows binding to the glucagon receptor. Indeed, it was later demonstrated that the protonatable histidine imidazole is important for transduction. It is likely that the interaction of a positively charged histidine 1 with a negatively charged aspartic acid 9 might be part of the triggering step at the molecular level. Two other aspartic acid residues in glucagon are capable of assuming a similar role, namely that of contributing to an electrostatic attraction with histidine via a negative carboxylate. These studies were conducted to investigate the role of aspartic acid 15 and 21 in glucagon action. Evidence reported here, gathered from 31 replacement analogs, supports the idea that in the absence of the requisite carboxyl group at position 9, histidine utilizes Asp21 or Asp15 as a compensatory site. Asp15 was also found to be indispensable for binding and may serve to tether the hormone to the receptor protein at the binding site. It is also demonstrated that these new findings promote the design of better glucagon antagonists.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine.

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1990

Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.

ADP Ribose Transferases↗
PubMed2000

Stereoselective synthesis of beta-benzyl-alpha-alkyl-beta-amino acids from L-aspartic acid.

A stereoselective synthesis of beta-benzyl-alpha-alkyl-beta-amino acids 1 and 2 from L-aspartic acid 3 has been developed. Methyl 5-phenyloxazolidin-2-one-4-acetate 4 was prepared from L-aspartic acid 3 through the acylation of benzene or phenyllithium with alpha-amino carboxyl group of L-aspartic acid skeleton. Alkylation of a dianion of 4 with alkyl halides and subsequent hydrogenation afforded anti-disubstituted beta-amino acids 1b and 1c in high stereoselectivities. Complete reversal of the stereoselection was realized by the alkylation of 4-phenyl-3-tert-butoxycarbonylamino-4-butanolide 6 which was obtained in a single step from 4. The 2,3,4-trisubstituted amino lactone 7 thus obtained was hydrogenated to give a syn-disubstituted beta-amino acid 2a. The syn-products 2b, 2c, and 2dwere alternatively prepared via aldol condensation of 6 with aromatic or aliphatic aldehydes followed by stereoselective reduction of the double bond with nickel chloride-sodium borohydride.

Acylation↗
PubMed2000

Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase.

Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.

Amino Acid Sequence↗
PubMed1994

A physiological role for N-methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptors in pulsatile gonadotropin secretion in the adult female rat.

The present study was designed to evaluate the physiological role of excitatory amino acids (EAAs) in the pulsatile secretion of LH and FSH. Specific antagonists for N-methyl-D-aspartic acid (NMDA) receptors and kainate/quisqualate (non-NMDA) receptors were used to achieve this aim. Adult female rats (250-280 g), ovariectomized for 2 weeks, were implanted with a cannula in the third ventricle of the brain. One week later, 2-amino-5-phosphono-pentanoic acid (AP-5; 10 micrograms/rat), a specific competitive NMDA receptor antagonist; 6,7-dinitroquinoxaline-2,3-dione (DNQX; 30 nM), a selective antagonist of non-NMDA receptors; or the same volume of saline, was injected via the third ventricle to conscious and unrestrained ovariectomized animals. Blood samples were collected from indwelling jugular catheters 20 min before and after injection and at 10-min intervals for 100 min for plasma LH and FSH determinations. The results revealed that the administration of either AP-5 or DNQX significantly suppressed mean as well as trough LH levels. This suppression was accompanied by a suppression of LH pulse frequency and LH pulse amplitude. AP-5 suppressed LH pulse amplitude, through LH levels, and mean LH levels to a greater degree than DNQX. The analysis of FSH pulses showed that AP-5 inhibited mean and trough FSH levels, and this appeared to be achieved by the suppression of FSH pulse amplitude, but not frequency. DNQX, on the other hand, did not significantly alter FSH pulse frequency, pulse amplitude, or trough or mean FSH levels. Taken as a whole, the present study provides evidence that endogenous EAAs, acting through both NMDA and non-NMDA receptors, play an important physiological role in the generation of pulsatile LH secretion in adult female rats. FSH pulse frequency was not significantly affected by the administration of either EAA receptor antagonist, although AP-5 did suppress FSH pulse amplitude and mean and trough FSH levels.

2-Amino-5-phosphonovalerate↗
PubMed2002

Improvement of the functions of osteoblasts seeded on modified poly(D,L-lactic acid) with poly(aspartic acid).

One of the challenges in the field of tissue engineering is the development of biomaterial/cell interactions. For the purposes of the present study, two molecular weights of poly(aspartic acid) (PASP) were used to modify poly(D,L-lactic acid) (PDLLA) films in order to enhance their cell affinity. The properties of the PDLLA-modified surfaces and the controls were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). These data reflect the change in the biocompatibility of modified PDLLA surfaces. Then rat osteoblasts were seeded onto these modified surfaces and on controls to examine their effects on cell adhesion and proliferation. Cell morphologies on these surfaces were studied by scanning electron microscopy (SEM), and cell viability was evaluated with a MTT assay. In addition, differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity. The results suggest that PASP-modified surfaces may enhance the interactions between osteoblasts and PDLLA films.

Alkaline Phosphatase↗
PubMed2000

The role of D-aspartic acid and N-methyl-D-aspartic acid in the regulation of prolactin release.

In this study, using an enzymatic HPLC method in combination with D-aspartate oxidase, we show that N-methyl-D-aspartate (NMDA) is present at nanomolar levels in rat nervous system and endocrine glands as a natural compound, and it is biosynthesized in vivo and in vitro. D-aspartate (D-Asp) is its natural precursor and also occurs as an endogenous compound. Among the endocrine glands, the highest quantities of D-Asp (78 +/- 12 nmol/g) and NMDA (8.4 +/- 1.2 nmol/g) occur in the adenohypophysis, whereas the hypothalamus represents the area of the nervous system where these amino acids are most abundant (55 +/- 9 and 5.6 +/- 1.1 nmol/g for D-Asp and NMDA, respectively). When D-Asp is administered to rats by ip injection, there is a significant uptake of D-Asp into the adenohypophysis and a significant increase in the concentration of NMDA in the adenohypophysis, hypothalamus and hippocampus, suggesting that D-Asp is an endogenous precursor for NMDA biosynthesis. Experiments conducted on tissue homogenates confirm that D-Asp is the precursor of the NMDA and that the enzyme catalyzing this reaction is a methyltransferase. S-adenosyl-L-methionine (SAM) is the methyl group donor. In vivo experiments consisting of ip injections of sodium D-aspartate show that this amino acid induced a significant serum PRL elevation and this effect is dose and time dependent. In vitro experiments conducted on isolated adenohypophysis or adenohypophysis coincubated with the hypothalamus, showed that the release of PRL is caused by a direct action of D-Asp on the pituitary gland and also mediated by the indirect action of NMDA on the hypothalamus. Then, the latter induces the release of a putative factor that in turn stimulates the adenohypophysis reinforcing the PRL release. In conclusion, our data suggest that D-Asp and NMDA are present endogenously in the rat and are involved in the modulation of PRL release.

Animals↗
PubMed2000

Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release.

Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.

Animals↗
PubMedDate not supplied

Pharmacokinetics of arginine and aspartic acid administered simultaneously in the rat--III: Changes in the levels of amino acids in the plasma, liver and brain after simultaneous administration of arginine and aspartic acid.

Changes in the levels of amino acids in the plasma, liver and brain were studied in rats after simultaneous administration of 0.1 mmol/rat of arginine and aspartic acid. The levels of most of the assayed amino acids underwent various changes some of which only occurred in one of the organs studied, for example, GABA in the brain and proline in the liver. It is difficult to advance any explanation. Meanwhile some of changes can be explained on the grounds of well-known metabolic changes. The large increase of ornithine in the liver may be due to the action of arginase in the first stage in the urea cycle. This amino acid is the precursor of glutamic acid and proline which may explain the high levels of these two amino acids observed especially in the liver. The increase of GABA in the brain may be due to the simultaneous administration of arginine and aspartic acid which could induce the formation of ornithine and alpha-ketoglutaric acid respectively, two metabolites known to increase cerebral GABA. The increases in Ser, Gly, and Ala observed in practically all the tissues studied may be due to the formation of oxaloacetate from aspartic acid.

Administration, Oral↗