Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ASPARTIC ACID”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources
PubMed2004

Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding.

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.

Acetylcholine↗
PubMed1993

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1.

The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.

Amino Acid Sequence↗
PubMed2006

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif.

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.

Adenosine Diphosphate↗
PubMed1994

Opioidergic modulation of N-methyl-D,L-aspartic-acid-stimulated LH release in young adult but not older male mice.

Among the neuromodulators of the GnRH neuronal system are the endogenous opioid peptides and the excitatory amino acids. Although the opioid antagonist naloxone (NAL) induces LH secretion in many species, there are no reports of an effect of NAL on LH release in mice. Our previous studies demonstrated that the excitatory amino acid analog N-methyl-D,L-aspartic acid (NMA) stimulates LH release in mice and suggested that NMA-induced LH release is mediated via afferents to GnRH neurons. In the current study, the role of the endogenous opioid system in the regulation of LH release in adult male mice was assessed by testing whether this system is a component of the NMA-stimulated LH response. NAL, its quaternary derivative NAL methiodide (which remains outside of the blood-brain barrier) and saline (SAL) were administered alone and in combination with NMA via intravenous catheters. Although neither opioid antagonist stimulated LH release when administered alone, each significantly potentiated the LH response to NMA in young adult (10- to 14-week-old) male mice (p < 0.01) but not in older (10- to 16-month-old) male mice. The equivalent action of the two ipioid blockers suggested an action outside of the blood-brain barrier. To determine whether opioid blockade altered pituitary sensitivity to GnRH, a dose response for exogenously administered GnRH was first determined, and low and high doses of GnRH were tested in combination with NAL or SAL. Neither treatment was effective in altering the LH response to GnRH, indicating that the action of the opioid antagonists was at a suprapituitary location. (ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1998

Identification of the aspartic acid residue located at or near substrate-binding site of rye seed chitinase-c.

Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) at pH 5.5 and 5 degrees C in the presence and absence of (GlcNAc)4. In the absence of (GlcNAc)4, 5.2 carboxyl groups were modified by 90 min-reaction and the chitinase activity was reduced to 2.0%, while in the presence of (GlcNAc)4, 4.6 carboxyl groups were modified and 72% of the activity was retained. To identify the carboxyl group protected by (GlcNAc)4 from the modification, RSC-c was first modified with EDC and GEE in the presence of (GlcNAc)4 and then radiolabeled with EDC and [14C]GEE in the absence of (GlcNAc)4. Analyses of the radioactive peptides from the tryptic and chymotryptic digests of radiolabeled RSC-c showed that the main radiolabeled carboxyl group is that of Asp95, suggesting that Asp95 is located at or near substrate-binding site of RSC-c.

Acetylglucosamine↗
PubMed1992

Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the Schiff base.

In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 +/- 0.1 and the apparent pK for protonation of the His85 side chain to be approximately 3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.

Amino Acid Sequence↗
PubMed1991

Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

Amino Acid Sequence↗
PubMed1991

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Amino Acid Sequence↗
PubMed1990

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism.

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.

Amino Acid Sequence↗