Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating a single internal serine residue of the initial enzyme in the biosynthetic pathway, prostaglandin synthetase. In this study, the region of the enzyme that is modified by aspirin has been isolated, and its amino acid sequence has been determined. Sheep vesicular gland [acetyl-3H]prostaglandin synthetase was purified following treatment with [acetyl-3H]aspirin and digest with pepsin. An acetyl-3H-labeled peptic peptide of approximately 25 residues was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined to be Ile-Glu-Met-Gly-Ala-Pro-Phe-Ser-Leu-Lys-Gly-Leu-Gly-Asn-Pro-Ile-Glu-Ser-Pro-Glu-Tyr. The acetylated serine residue was located at position 8 in this sequence. The current study marks this polypeptide sequence as a region related to an active site of the enzyme.     

Human lamin B contains a farnesylated cysteine residue

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.     

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta-bromoacetoxydihydrotestosterone

This article deals with the elucidation of the steroid-binding site of human sex hormone-binding globulin (SHBG). 17 beta-Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in a time-dependent and irreversible fashion. The interaction could be totally inhibited by the simultaneous addition of an excess of dihydrotesterone. At the completion of the reaction, the molar ratio of BA-DHT to SHBG was approximately unity. SHBG was affinity labeled with [14C]BA-DHT and submitted to acid hydrolysis. The released amino acids were evaluated on high performance liquid chromatography, and virtually all of the 14C was identified as 3-[14C]carboxymethylhistidine. Furthermore, [14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation of the released tryptic peptides by reverse-phase high performance liquid chromatography. The 14C was localized to a single tryptic peptide. It contained 2' histidyl residues, corresponding to residues 235 and 251 in the known amino acid sequence of SHBG. Although most of the 3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was trapped on the filter of the amino acid sequenator, sufficient radioactivity emerged to identify histidyl residue 235 as the labeled amino acid.     

Polyamines in vaccinia virions and polypeptides released from viral cores by acid extraction.

Vaccinia virions propagated in the presence of [3H]ornithine were found to contain two labeled polyamines, spermine and spermidine. In complete virions the ratio of radioactively labeled spermine to spermidine was about 1:10, whereas in viral cores the ratio was 2:5. This suggests that some spermidine was preferentially lost during the conversion of virions to cores or that spermidine was present in the virions both inside and outside the core structure. Addition of [3H]ornithine to vaccinia virus-infected cells as late as 6 h postinfection demonstrated that, although the conversion of this precursor to polyamines was reduced by 50% or more as compared to mock-infected cells, complete inhibition of polyamine synthesis did not occur. Two percent or less of the total radioactivity associated with virions grown in the presence of [3H]ornithine was found to be acid soluble. Polyacrylamide gel electrophoretic analysis showed that all the structural polypeptides were labeled when virions were propagated in the presence of [3H]ornithine. When cores labeled with a mixture of 14C-labeled amino acids were extracted with 0.25 N H2SO4, 12 to 15% of the labeled core polypeptides were released and could be precipitated with acetone. About 40% of [3H]arginine-labeled polypeptides associated with cores were extracted with acid. Four polypeptides or groups of polypeptides were resolved after polyacrylamide gel electrophoresis analysis of the acid-soluble fraction of cores with molecular weights of about 58,000, 34,000, 24,000 and 10,000 to 12,000. About 40% of the [3H]arginine radioactivity extracted from cores coelectrophoresed with the 10,000 to 12,000-molecular weight polypeptide, indicating that this may represent an arginine-rich, histone-like structural polypeptide of the virion.     

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride

Further chemical evidence has been obtained using NaB3H4 to support our previous assignment of a thiol ester bond in human C3 (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768). Following trypsin activation of human C3 in the presence of NaB3H4, 3H was shown to have incorporated specifically into the alpha'-chain of C3b. Subsequent fragmentation of [3H]C3b with porcine elastase further localized the label to the C3d subdomain. Under identical conditions, native C3 or C3 pretreated with trypsin (C3b) showed low reactivity with NaB3H4. A tryptic peptide containing the 3H label was isolated following digestion of [3H]C3b on activated thiol-Sepharose. After hydrolysis and saponification of the peptide hydrolysate, amino acid analysis indicated that the 3H had been incorporated into alpha-amino-delta-hydroxyvaleric acid, the product expected from reduction of an ester bond involving a glutamyl residue. On sequence analysis of the labeled peptide, the 3H was shown to reside at the position of the glutamyl residue previously proposed to be involved in the thiol ester bond. The residue at this position was confirmed as alpha-amino-delta-[3H] hydroxyvaleric acid by high performance liquid chromatography analysis and, after back hydrolysis, by amino acid analysis. These data significantly strengthen earlier studies which indicated the presence of a beta-Cys-gamma-Glu thiol ester bond in human C3.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene

A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, has a high mannose and a modified (fucosylated) oligosaccharide on each polypeptide. Fractionation by high performance liquid chromatography of tryptic digests of [³H]fucose or [³H]glucosamine labeled phytohemagglutinin, followed by amino acid sequencing of the isolated glycopeptides, shows that the high mannose oligosaccharide is attached to Asn¹² and the modified oligosaccharide to Asn⁶⁰ of the protein. In animal glycoproteins, high mannose chains are rarely found at the N-terminal side of complex chains.     

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.     

Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3

The proteolipid subunit of the ATPase complex was identified in whole membranes of the thermophilic bacterium PS-3 by means of a covalent modification with the 14C-labelled inhibitor dicyclohexylcarbodiimide. The proteolipid could be purified from the membrane in free and carbodiimide-modified form by extraction with chloroform/methanol and subsequent carboxymethylcellulose chromatography in mixtures of chloroform/methanol/water. The complete amino acid sequence of the 72-residue polypeptide could be determined by automated solid-phase Edman degradation of the whole protein, and of fragments obtained after cleavage with cyanogen bromide and N-bromosuccinimide. Chemical cleavages and separations of the resulting fragments by gel chromatography were performed in 80% formic acid. The amino acid sequence shows a concentration of hydrophobic amino acids in two segments of about 25 residues at the amino-terminal and carboxy-terminal ends. The polar residues are clustered in the middle of the polypeptide chain. The bound [14C]dicyclohexylcarbodiimide label is recovered exclusively at position 56, which is occupied by a glutamyl residue. The proteolipid from PS-3 exhibits homology to the corresponding ATPase subunit from mitochondria. The carbodiimide-reactive glutamyl residue occurs at the position as in the mitochondrial proteins.     

Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from the purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine-labeled from purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.     

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.     

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the NAD (H)--binding region of the mitochondrial nicotinamide nucleotide transhydrogenase modified by N,N'-dicyclohexylcarbodiimide

Purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and NAD(H) protects the enzyme against this inhibition [Phelps, D.C., and Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The tryptic digest of TH treated with [14C]DCCD showed a single radioactive peak upon FPLC chromatography. This radioactive peak was absent from tryptic digests of TH treated with [14C]DCCD in the presence of NADH. Sequence analysis of the radioactive peak showed that it contained two peptides, one derived from the other as a result of incomplete cleavage by trypsin of a lysyl-glutamyl bond. After further digestion with Staphylococcus V8 protease, the smaller radioactive fragment was isolated and sequenced. The amino acid sequence of this fragment, as determined by manual Edman degradation, was Ala-Glu-Met-Lys. The second residue was modified. Amino acid analysis and sequence studies on the radioactive tryptic peptide mixture indicated that the sequence around the DCCD-modified residue was Glu-Met-Ser-Lys-Glu-Phe-Ile-Glu-Ala-Glu-Met-Lys. In other studies, this sequence has been found in the amino acid sequence of TH as predicted from the corresponding cDNA. The DCCD-modified peptide is near the site of NAD(H) binding, as labeled with radioactive p-fluorosulfonylbenzoyl-5'-adenosine. Furthermore, there is a high degree of homology in this region between the amino acid sequences of the bovine heart TH and the alpha subunit of the Escherichia coli TH.