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PubMed1992

The active site of creatine kinase. Affinity labeling of cysteine 282 with N-(2,3-epoxypropyl)-N-amidinoglycine.

Epoxycreatine (N-(2,3-epoxypropyl)-N-amidinoglycine) is an affinity label of creatine kinase that irreversibly and completely inactivates the enzyme (Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 254, 1879-1886). To identify active site residues of rabbit muscle creatine kinase, the site of modification of it by epoxycreatine has been determined. Separation by high performance liquid chromatography of a tryptic digest of [14C]epoxycreatine-modified creatine kinase yielded two radiolabeled peptides. The larger of these consisted of amino acids Ala-266 through Arg-291 and was labeled with epoxycreatine at Cys-282. Attempts to purify completely the other labeled peptide were not successful; however, it was possible to obtain, by tandem mass spectrometry, a collision-induced dissociation spectrum of it from a mixture of several peptides. This peptide was a fragment (amino acids Val-279 through Arg-291) of the previously identified peptide and was also labeled at Cys-282. Model studies with cysteine and epoxycreatine have demonstrated that opening of the oxirane ring occurs by attack of the cysteine thiolate at the terminal carbon of the epoxide. These results are consistent with previous studies on the base lability of the label; however, a carboxyl group in the active site is not labeled, as had been previously suggested. These results provide evidence that Cys-282 is located in or near the creatine-binding site and will also be important in identifying and delineating the boundaries of the active site of creatine kinase.

Affinity Labels↗

PubMed1992

m-Sulfonate benzene diazonium chloride: a powerful affinity label for the gamma-aminobutyric acid binding site from rat brain.

m-Sulfonate benzene diazonium chloride (MSBD) was used to affinity-label the gamma-aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter-adsorbed membranes. Inactivation of specific [3H]-muscimol binding sites by MSBD and its prevention by GABA were both time- and concentration-dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.

Affinity Labels↗

PubMed1999

New opioid affinity labels containing maleoyl moiety.

Opioid receptor binding properties and pharmacological profiles of novel peptides containing maleoyl function were determined in order to develop new affinity labels. Based on the enkephalin structure peptide ligands were synthesized and tested. Both in in vitro receptor binding experiments and pharmacological studies, all ligands showed agonist character with relatively high affinity (Ki values in the nanomolar range) and good to moderate selectivity. Replacement of Gly2 in the enkephalin frame with D-Ala led to higher affinities with a small decrease in selectivity. The longer peptide chains resulted in compounds with high percentage (up to 86%) of irreversible binding. The selectivity pattern of the ligands is in good agreement with the data obtained from the pharmacological assays (guinea pig ileum and mouse vas deferens bioassays). The newly synthesized peptides could be used in further studies in order to determine more detailed characteristics of the ligand-receptor interaction.

Affinity Labels↗

PubMed1978

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase.

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.

Affinity Labels↗

PubMed1983

Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.

2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.

Affinity Labels↗

PubMed1988

Affinity labels for the anion-binding site in ovotransferrin.

Bromopyruvate, a known alkylating agent, has previously been reported to function as an affinity label for the anion-binding site in the iron-binding protein ovotransferrin [Patch, M.G., & Carrano, C. J. (1982) Biochim. Biophys. Acta 700, 217-220]. However, the present results indicate that hydroxypyruvate also functions in an almost identical manner, which implies that alkylation of a susceptible nucleophile cannot be the mechanism responsible for the covalent attachment of the anion. Model complexes and amino acid analysis of labeled ovotransferrin suggest that initial Schiff base formation, followed by reduction of the imine bond between the affinity anion and a lysine within the locus of the anion-binding site, accounts for the irreversible labeling. As expected, the covalently attached anions render the iron in the ovotransferrin-iron-anion ternary complex much more resistant to loss at low pH. It is proposed that the covalently labeled protein be used to test the hypothesis that iron removal from transferrin occurs by protonation and loss of the anion in low-pH lysosomal vesicles.

Affinity Labels↗

PubMed2000

Protein-carbohydrate interactions in human lysozyme probed by combining site-directed mutagenesis and affinity labeling.

The synergism between apolar and polar interactions in the carbohydrate recognition by human lysozyme (HL) was probed by site-directed mutagenesis and affinity labeling. The three-dimensional structures of the Tyr63-->Leu mutant HL labeled with 2',3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose (L63-HL/NAG-NAG-EPO complex) and the Asp102-->Glu mutant HL labeled with the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine were revealed by X-ray diffraction at 2.23 and 1.96 A resolution, respectively. Compared to the wild-type HL labeled with the 2', 3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose, the N-acetylglucosamine residue at subsite B of the L63-HL/NAG-NAG-EPO complex markedly moved away from the 63rd residue, with substantial loss of hydrogen-bonding interactions. Evidently, the stacking interaction with the aromatic side chain of Tyr63 is essential in positioning the N-acetylglucosamine residue in the productive binding mode. On the other hand, the position of the galactose residue in subsite B of HL is almost unchanged by the mutation of Asp102 to Glu. Most hydrogen bonds, including the one between the carboxylate group of Glu102 and the axial 4-OH group of the galactose residue, were maintained by local movement of the backbone from residues 102-104. In both structures, the conformation of the disaccharide was conserved, reflecting an intrinsic conformational rigidity of the disaccharides. The structural analysis suggested that CH-pi interactions played an important role in the recognition of the carbohydrate residue at subsite B of HL.

Affinity Labels↗

PubMed1987

Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate.

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.

Acetamides↗

PubMed1982

Affinity labeling of purified ornithine decarboxylase by alpha-difluoromethylornithine.

Ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) purified from rat liver was affinity-labeled by alpha-[5-14C]difluoromethylornithine. On analysis by SDS-polyacrylamide gel electrophoresis, the radioactivity migrated as a single major peak that coincided with a single protein band of Mr 50,000. Calculation from bound radioactivity indicated that ornithine decarboxylase has two active sites, one for each subunit, and that pure enzyme should have a specific activity of about 1.4 x 10(6) nmol CO2/h per mg protein.

Affinity Labels↗

PubMed1988

Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP.

gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.

Affinity Labels↗

PubMed1981

Potential thyroliberin affinity labels. 1. Chloroacetyl-substituted phenylalanylpyrrolidines.

Six analogues of thyroliberin (TRH) that have a chloroacetyl substituent at the amino terminus have been prepared as potential affinity labels for the TRH receptor. These compounds are N-(chloroacetyl)-L-alanyl-L-phenylalanylpyrrolidine (ClAc-Ala-Phe-Pyrr; 14), N-[m-(chloroacetyl)benzoyl]-L-phenylalanylpyrrolidine (m-ClAcBz-Phe-Pyrr; 11a), N-[m-(chloroacetyl)benzoyl]-L-alanyl-L-phenylalanylpyrrolidine (m-ClAcBz-Ala-Phe-Pyrr; 15a), N-[p-(chloroacetyl)benzoyl]-L-phenylalanylpyrrolidine (p-ClAcBz-Phe-Pyrr; 11b), and N-[p-(chloroacetyl)benzoyl]-L-alanyl-L-phenylalanylpyrrolidine (p-ClAcBz-Ala-Phe-Pyrr; 15b). Pyroglutamyl-L-phenylalanylpyrrolidine was also synthesized as a model agonist. Weak agonist activity was observed for 11a, 11b, and 15b. These three analogues do not contain the amide group of the pyroglutamyl moiety that was previously thought to be essential for intrinsic activity. No significant antagonist activity was observed for these compounds at the doses tested.

Affinity Labels↗

PubMed2004

S-(2,3-dichlorotriazinyl)glutathione. A new affinity label for probing the structure and function of glutathione transferases.

S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 +/- 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme (K(d) 107.9 +/- 2.1 micro m for the fast phase, and 224.5 +/- 4.2 micro m for the slow phase) before irreversible modification with maximum rate constants of 0.049 +/- 0.002 min(-1) and 0.0153 +/- 0.001 min(-1) for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), approximately 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of alpha-helix H"'(3) and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites.

Affinity Labels↗

PubMed1986

Characterization of benzodiazepine binding sites after short-wave photo-affinity labeling with flunitrazepam.

Membranes prepared from rat cerebral cortex were irradiated with short-wave UV light in the presence of flunitrazepam (FZ). This photo-affinity labeling (PAL) drastically reduces the potency of FZ binding to these membranes, but the binding of 3H-beta-carboline-3-carboxylate ethyl ester (3H-BCCE) was found to be essentially unchanged. 3H-BCCE binding was therefore determined in the presence of an antagonist (BCCE itself), an agonist (FZ) and a compound reported to discriminate between multiple benzodiazepine sites (CL 218,872). The results with BCCE are consistent with a single population of sites, but FZ binds to some of the sites with a reduced affinity (KI = 30 nM) and to the remaining sites with a very low affinity (KI approximately equal to 1 microM). CL 218,872 shows a reduced affinity but appears to interact with all of the sites. Taken together, these results indicate that the binding domains for BCCE and FZ are not identical, and that CL 218,872 interacts more strongly with the antagonist domain.

Affinity Labels↗

PubMed1976

Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts.

The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.

Adenosine Triphosphate↗

PubMed1980

Photo-induced affinity labeling of Escherichia coli ribosomes by chloramphenicol.

In order to obtain more information about the binding site for chloramphenicol (D-threo diastereoisomer) on the bacterial ribosome, photo-affinity labeling experiments of this receptor have been performed with [3H]chloramphenicol itself. Control experiments show that this drug can be split photochemically by ultraviolet irradiation, whereas the ribosome is not modified structurally or functionally by such a treatment. When photolysis of a mixture of chloramphenicol and ribosomes is performed under critical conditions, some proteins like L1, L11, S3 and S4 are radiolabeled. L11, S3 and S4 are radiolabeled specifically as demonstrated by photo-incorporation experiments with isotopically diluted [3H]chloramphenicol or by comparison of the results obtained here with reversible experiments performed by the isotopic dilution method. When the D-erythro diastereoisomer of chloramphenicol is photo-incorporated into the bacterial ribosome, proteins are radiolabeled only in a non-specific way. These results show that this material could be used as an efficient scavenger. When finally D-threo [3H]chloramphenicol is photo-incorporated in the presence of a large amount of the D-erythro diastereoisomer, the radiolabeling pattern obtained for the proteins is quite different from that expected: while L11 is still labeled fairly extensively, L27 is the most radiolabeled protein found.

Affinity Labels↗

PubMed1992

Human immunodeficiency virus type 1 reverse transcriptase. Affinity labeling of the primer binding site.

Affinity modification of the primer site of HIV1-RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5' end of d(pT)10. The affinity of HIV1-RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV-irradiation at 303-365 nm in the presence of ArN3-d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the Km value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNA(Lys).

Affinity Labels↗

PubMedDate not supplied

A comparison of 11 beta-chloromethylestradiol and tamoxifen aziridine as affinity labeling reagents for estrogen receptors.

The tritium-labeled from of 11 beta-chloromethylestradiol was prepared by metal hydride reduction of the 17-keto derivative. Affinity labeling experiments were carried out using [3H] 11 beta-chloromethylestradiol and [3H]tamoxifen aziridine with estrogen receptor from crude, calf uterine cytosol and partially purified (heparin-sepharose chromatography) preparations. Both compounds formed highly stable receptor complexes. Estrogen specific, covalent binding, however, was indicated only for [3H]tamoxifen aziridine. An equilibrium dissociation constant of 2.8 x 10(-10) M was determined for the receptor-[3H] 11 beta-chloromethylestradiol interaction. Measurement of hormone dissociation kinetics at 30 degrees C revealed a slow, single phase dissociation of 11 beta-chloromethylestradiol from the receptor (dissociation rate constant, 1.3 x 10(-3) min-1). This contrasted with the normal biphasic dissociation pattern of estradiol in which the dissociation rate constant for the slower component was 16.7 x 10(-3) min-1. The results indicate that 11 beta-chloromethylestradiol readily converts the estrogen receptor to a high affinity binding form and suggest that the radiolabeled hormone may prove useful for studies of estrogen action.

Affinity Labels↗

PubMed1987

Ketononestrol aziridine, an agonistic estrogen receptor affinity label: study of its bioactivity and estrogen receptor covalent labeling.

Ketononestrol aziridine [(6R,TS)1-(N-aziridinyl)6,7-bis-(4-hydroxyphenyl)5-nonamone (KNA)], an aziridine derivative of hexestrol, is an estrogenic affinity label for the estrogen receptor (ER). It has an apparent relative binding affinity 8% that of estradiol and shows time-dependent irreversible binding to the ER in uterine cytosol preparations and intact human breast cancer cells (MCF-7). The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases the cell growth rate and elevates the level of progesterone receptor. KNA was prepared in high specific activity tritium-labeled form by iodination of a methanesulfonate precursor, followed by catalytic tritium-iodine exchange and aziridinylation; the material prepared has high radiochemical purity and a specific activity of 67 Ci/mmol. The covalent attachment of [3H]KNA to the ER can be followed directly by a solvent precipitation assay. In cytosol preparations of uterine ER, labeling with [3H]KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER in intact MCF-7 cells can also be covalently labeled by treatment with [3H]KNA. Receptor covalently labeled with [3H]KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antireceptor antibodies. On sodium dodecyl sulfate-polyacrylamide gels, the principal labeled species migrates with a mol wt of 66,000. KNA should prove to be a useful probe for studies on receptor structure, function, and chromatin interactions, particularly when the behavior of a receptor-agonist complex is being investigated.

Affinity Labels↗