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PubMed1978

Side reactions in peptide synthesis. VI. A reexamination of the benzyl group in the protection of the side chains of tyrosine and aspartic acid.

The acid catalyzed O leads to C migration of the benzyl group in the side chain of tyrosine could be reduced by applying HBr in a mixture of phenol and p-cresol instead of BHr in trifluoroacetic acid for acidolytic deprotection. This side reaction occurs also during the removal of Boc groups. The loss of O-benzyl protection and the formation of 3-benzyltyrosine residues could be suppressed by the application of a 7:3 mixture of trifluoroacetic acid and acetic acid. The acid- and base-catalyzed ring closure of beta-benzylaspartyl residues to aminosuccinyl derivatives was also studied. In this case HBr in trifluoroacetic acid was found to be relatively harmless. Deprotection with HBr in a mixture of trifluoroacetic acid and p-cresol can be applied for peptides that contain both beta-benzylaspartyl and O-benzyltyrosyl residues. An attempt to reduce the rate of the base-catalyzed side reaction by application of hindered tertiary amines was abandoned because the tertiary amines which were effective in this respect let to significant reduction of the rate of the desired reaction, the aminolysis of active esters, as well. A satisfactory solution for the problem was found in the selective catalysis of the active ester reaction with 1-hydroxybenzotriazole or 4-dimethyl-aminopyridine. These catalysts do not enhance the rate of ring closure and in their presence essentially pure beta-benzylaspartyl peptides can be produced in good yield.

Aspartic Acid↗
PubMed2003

A dual role for an aspartic acid in glycosylasparaginase autoproteolysis.

Glycosylasparaginase uses an autoproteolytic processing mechanism, through an N-O acyl shift, to generate a mature/active enzyme from a single-chain precursor. Structures of glycosylasparaginase precursors in complex with a glycine inhibitor have revealed the backbone in the immediate vicinity of the scissile peptide bond to be in a distorted trans conformation, which is believed to be the driving force for the N-O acyl shift to break the peptide bond. Here we report the effects of point mutation D151N. In addition to the loss of the base essential in autoproteolysis, this mutation also eradicates the backbone distortion near the scissile peptide bond. Binding of the glycine inhibitor to the autoproteolytic site of the D151N mutant does not restore the backbone distortion. Therefore, Asp151 plays a dual role, acting as the general base to activate the nucleophile and holding the distorted trans conformation that is critical for initiating an N-O acyl shift.

Aspartic Acid↗
PubMed1992

The phosphorylation site of the Kdp-ATPase of Escherichia coli: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit.

The potassium-translocating Kdp-ATPase of Escherichia coli shares common functional properties with eukaryotic P-type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp-307 in KdpB by Glu, Asn, Gln, Tyr, His, Ala or Ser by site-directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K+ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp-307 in KdpB is the phosphorylation site of the Kdp-ATPase. In contrast, replacement of the close but non-conserved Asp-300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+ of the ATPase activity has been increased 30-fold compared with the wild-type enzyme.

Adenosine Triphosphatases↗
PubMed1988

Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212.

Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.

Aspartic Acid↗
PubMed1998

An aspartic acid residue near the second transmembrane segment of ATP receptor/channel regulates agonist sensitivity.

Charged or polarized amino acid residues near or within the second transmembrane (M2) segment of neuronal ATP receptor/channels (P2X2 receptors) were neutralized by site-directed mutagenesis, and the properties of the mutants were electrophysiologically characterized using Xenopus oocytes. When Asp315 was substituted with Val (D315V), the sensitivity to ATP was reduced by about 60-fold. The sensitivity to ATP was not affected by the neutralization of Lys324, which is involved in a Walker type A ATP-binding sequence, Lys366, Tyr330, or Asn333. With D315V channels, the sensitivities to other agonists (ADP, ATP gamma S, and 2-methylthio ATP) were also reduced. The sensitivities to antagonists (suramin and Cibacron Blue F3GA) were, however, not affected by this neutralization. The results suggest that Asp315, which is assumed to be present in the extracellular region near the M2 segment of P2X2 receptor/channels, serves to maintain agonist sensitivity.

Adenine Nucleotides↗
PubMed2005

Roles of the threonine 407, aspartic acid 417, and threonine 419 residues in P450 2B1 in metabolism.

We have previously observed that the quadruple (S407T-N417D-A419T-K473M) and triple (S407T-N17D-A419T) mutants of the chimeric construct of P450 2B1/2B2 do not undergo mechanism-based inactivation by 17alpha-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether (tBMP). The ability of these mutants to metabolize 17EE, benzphetamine, and testosterone has been investigated. The profile for 17EE metabolism by both mutants was characteristic of both wild-types. The two mutants metabolized testosterone to form androstenedione with no formation of the hydroxy products as was seen with both the wild-types. Benzphetamine metabolism by the mutants showed that both mutants exhibited an increased tendency to catalyze demethylation rather than debenzylation. In the presence of the alternate oxidants cumene hydroperoxide and tert-butyl hydroperoxide, the wild-type 2B1 was not inactivated by 17EE. Metabolism of 17EE by 2B1 supported by these alternate oxidants revealed differences in the metabolites that may be related to the inability of 2B1 to be inactivated under these conditions.

Aspartic Acid↗
PubMed1988

Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

Site-specific mutagenesis of the blood vessel-inducing protein angiogenin has been used to further explore both its homology to pancreatic ribonuclease and the functional roles of particular residues. Replacement of Asp-116 in angiogenin by either asparagine (D116N), alanine (D116A), or histidine (D116H) markedly enhances both its ribonucleolytic activity and angiogenic potency. Activity toward tRNA is 8-, 15-, and 18-fold greater than native angiogenin for D116N-, D116A-, and D116H-angiogenin, respectively. The enzymatic specificity of angiogenin, however, has been maintained. Thus, cleavage of 18S and 28S rRNA by the most active His-116 mutant yields the same pattern of polynucleotide products as from angiogenin, whereas there are only minor alterations in activity with cytidylyl(3',5')adenosine and uridylyl(3',5')-adenosine. Extensive biological assays on the chicken embryo chorioallantoic membrane demonstrate that D116H-angiogenin is one to two orders of magnitude more potent in inducing neovascularization than native angiogenin, which correlates well with enhanced enzymatic action. These results support the proposition that the enzymatic and angiogenic activities on angiogenin are interrelated.

Alanine↗
PubMed1981

Developmental changes of N-acetyl-L-aspartic acid, N-acetyl-alpha-aspartylglutamic acid and beta-citryl-L-glutamic acid in different brain regions and spinal cords of rat and guinea pig.

The developmental changes of N-acetylaspartic acid (NA-Asp), N-acetyl-alpha-aspartylglutamic acid (NA-Asp-Glu), and beta-citryl-L-glutamic acid (beta-CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of beta-CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and beta-CG content decreased to an adult level at birth.

Aging↗
PubMed1991

1H-NMR study of the intramolecular interaction of a substrate analogue covalently attached to aspartic acid-101 in lysozyme.

We prepared the lysozyme derivative in which the beta-carboxyl group of Asp101 was modified with alpha-O-methyl N-glycylglucosaminide as an amide by means of the carbodimide reaction (alpha-MGG lysozyme). Since Asp101 residue is located at the edge of the active site cleft, a 1H-NMR study was carried out for this derivative in order to investigate the interaction between the introduced substituent and the active site cleft. It was confirmed that the alpha-MGG moiety sat in the active site cleft in alpha-MGG lysozyme from the reduction of line broadening of the NH-proton of Trp63 located in the active site cleft, the remarkable chemical shift change of the methyl group of the alpha-MGG moiety upon adding a trimer of N-acetyl-D-glucosamine [(NAG)3], and the NOE between the C6-proton resonance of Trp63 and the methyl resonance of the alpha-MGG moiety. Furthermore, alpha-MGG lysozyme had increased thermal stability compared with native lysozyme. Therefore, it was concluded that the alpha-MGG moiety covalently attached to Asp101 interacted with the active site cleft to increase the thermal stability of lysozyme.

Animals↗
PubMed2001

Dehydration is catalyzed by glutamate-136 and aspartic acid-135 active site residues in Escherichia coli dTDP-glucose 4,6-dehydratase.

The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and rereduction. The enzyme contains the tightly bound coenzyme NAD(+), which mediates the dehydrogenation and rereduction steps of the reaction mechanism. In this study, we have determined that Asp135 and Glu136 are the acid and base catalysts, respectively, of the dehydration step. Identification of the acid catalyst was performed using an alternative substrate, dTDP-6-fluoro-6-deoxyglucose (dTDP-6FGlc), which undergoes fluoride ion elimination instead of dehydration, and thus does not require protonation of the leaving group. The steady-state rate of conversion of dTDP-6FGlc to dTDP-4-keto-6-deoxyglucose by each Asp135 variant was identical to that of wt, in contrast to turnover using dTDP-glucose where differences in rates of up to 2 orders of magnitude were observed. These results demonstrate Asp135's role in protonating the glucosyl-C6(OH) during dehydration. The base catalyst was identified using a previously uncharacterized, enzyme-catalyzed glucosyl-C5 hydrogen-solvent exchange reaction of product, dTDP-4-keto-6-deoxyglucose. Base catalysis of this exchange reaction is analogous to that occurring at C5 during the dehydration step of net catalysis. Thus, the decrease in the rate of catalysis ( approximately 2 orders of magnitude) of the exchange reaction observed with Glu136 variants demonstrates this residue's importance in base catalysis of dehydration.

Aspartic Acid↗
PubMedDate not supplied

[Synthesis of glutaminic and aspartic acids in mitochondria of the visual analyzer of the dog brain during postnatal ontogenesis].

It is revealed that glutaminic acid (GA) synthesis by direct reductive amination proves to be the most rapid in the mitochondria of the outer geniculate body (OGB) from the 1st till the 45th day of the postnatal development, and beginning from the 45th day till the 3-month age in visual cortex (field 17) (VC) and bigeminal bodies (BB). It is stated that GA synthesis by transamination in the mitochondria of VC, OGB, and BB proceeds less intensively than by a direct reductive amination at the early stage of postnatal development, being more rapid during the period from the 12-16th till the 45th day of postnatal development. Unlike GA synthesis in the mitochondria of the studied visual analyzer structures of canine brain AsA synthesis is more rapid in transamination reaction at the early stage of postnatal development. The high level of AsA synthesis was observed on the day when the puppies' eyes began to see clearly. The level of AsA synthesis by reductive amination and transamination in the mitochondria of VC, BB, and OGB at the early stage of postnatal development is lower than that of GA synthesis.

Aging↗
PubMed1981

Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases: neutron structure of trypsin.

A neutron structure analysis at 2.2-A resolution has been performed on bovine trypsin covalently inhibited by a transition-state analogue, the monoisopropylphosphoryl (MIP) group. The unique ability of neutron diffraction to locate hydrogen atoms experimentally has allowed the determination of the protonation states of the catalytic site residues (Asp-102 and His-57). Since the bound MIP group mimics the tetrahedral intermediate structure, these correspond to the protonation states at the most crucial step of the hydrolysis. This has resolved a much debated mechanistic issue by showing conclusively that the catalytic base in the transition state of the reaction is His-57, not Asp-102. This finding has important implications for the understanding of the hydrolysis mechanism of the serine proteases. A detailed examination of the stereochemical interaction among the catalytic groups was also conducted to identify their individual roles in the mechanism. Besides functioning as the catalytic group, it was found that His-57 could effectively "steer" the attacking water toward the acyl group during deacylation. Other aspects of protein structure which are observable only by neutron diffraction analysis are also discussed. These include orientation of well-ordered amide side chains, which is made possible by the large scattering difference between nitrogen and oxygen atoms, location and orientation of water molecules, and hydrogen exchange properties of the protein.

Aspartic Acid↗
PubMed1999

Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site interact with the penultimate aspartic acid and the C-terminal amide of cholecystokinin.

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR.CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.

Amides↗
PubMed1996

Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.

In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme.

Animals↗
PubMed1999

Mutagenesis of three residues, isoleucine-60, threonine-61, and aspartic acid-80, implicated in the GTPase activity of Escherichia coli elongation factor Tu.

The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.

Aspartic Acid↗
PubMed2002

Zinc-coordination of aspartic acid-76 in Sulfolobus ferredoxin is not required for thermal stability of the molecule.

The highly thermostable 7Fe-ferredoxin from Sulfolobus sp. strain 7 has tightly bound zinc at the interface between the N-terminal extra domain and the C-terminal core. The zinc is tetrahedrally ligated by His-16, His-19, His-34, and Asp-76. Previous studies on truncated mutants have shown that the zinc and certain parts, i.e. not all, of the N-terminal extra stretch are responsible for the thermal stabilization of the molecule. To study the role of Asp-76, a series of mutants were constructed with Asp-76 replaced by Glu (D76E), Asn (D76N), or Ala (D76A). All the mutants, as well as wild type ferredoxin, bound 1 mol zinc/mol protein, and showed similar kinetics for 2-oxoacid:ferredoxin oxidoreductase. The stability of the protein was examined by thermal degradation of the clusters. In the absence of guanidium thiocyanate, the T(m), defined as the mid-point temperature of the thermal transition from the native to the denatured state, for every mutant was above 100 degrees C. The T(m) values in the presence of 1 M guanidium thiocyanate were determined to be 90.8, 90.2, 87.1, 84.4, and 72.9 degrees C for the natural, recombinant, D76N-, D76A-, and D76E-ferredoxins, respectively. These results indicate that the interaction between zinc and the carboxyl oxygen of Asp-76 has subtle effects on both the zinc-ligation and stability, although the native zinc center is liganded with high symmetry, suggesting that the three His residues are more important for zinc-binding.

Aspartic Acid↗
PubMed2006

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin.

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.

Aspartic Acid↗
PubMed1995

Mutational analysis of the roles in catalysis and substrate recognition of arginines 54 and 305, aspartic acid 309, and tryptophan 317 located at subsites 1 and 2 in glucoamylase from Aspergillus niger.

The mutants Arg54-->Leu, Arg54-->Lys, Arg305-->Lys, Asp309-->Glu, and Trp317-->Phe, located at subsites 1 and 2 in glucoamylase from Aspergillus niger, provide insight into the importance of specific hydrogen bonds and hydrophobic interactions in substrate recognition, catalytic mechanism, and stability. As suggested from the crystal structure of a closely related glucoamylase [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639], Arg54 in subsite 1 hydrogen bonds to the key polar group 4'-OH of maltose. The two mutants of Arg54 display losses in transition-state stabilization of 16-21 kJ mol-1 in the hydrolysis of different maltooligodextrins, which originate from a [(1.2-1.8) x 10(3)]-fold reduction in kcat and changes in Km ranging from 25% to 300% of the wild-type values. Arg305 similarly hydrogen bonds to 2'-OH and 3-OH, located at subsites 1 and 2, respectively. Arg305-->Lys glucoamylase is not saturated at concentrations of maltose or maltoheptaose of 400- and 40-fold, respectively, the Km of the wild-type enzyme. This mutant also has highly reduced kcat. On the other hand, for the alpha-1,6-linked isomaltose, the Lys305 mutant surprisingly has the same Km as the wild-type enzyme, while kcat is 10(3)-fold reduced. Arg305 is thus an important determinant in the distinction of the alpha-1,4 to alpha-1,6 substrate specificity. Arg305 interacts electrostatically and hydrophobically with the side chains of Asp309 and Trp317.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗