Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

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.     

Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.     

Characterization of the binding of S-adenosyl-L-methionine to plasma membranes of HL-60 promyelocytic leukemia cells

S-Adenosyl-L-methionine (AdoMet) has been found to bind specifically to the plasma membrane of promyelocytic leukemia cells, HL-60. The Kd for AdoMet is 4.2.10(-6) M and the Bmax is 4.0.10(-12) mol/10(7) HL-60 cells. The binding is not related to the adenosine receptor since neither adenosine, ADP, nor ATP affect the ligand-receptor reaction. When HL-60 cells were incubated with physiological concentrations of [methyl-3H]AdoMet (20 microM) at 36 degrees C, AdoMet did not equilibrate with the intracellular pool, nor were any [3H]methyl groups incorporated into nucleic acids or proteins. In contrast, significant amounts of [3H]methyl groups were incorporated into membrane phospholipids. When cells were incubated with 20 microM [methyl-3H]AdoMet, [3H]methyl groups were transferred to phosphatidylethanolamine, -monomethylethanolamine, and -dimethylethanolamine yielding phosphatidylcholine. However, the rate of methyl transfer with AdoMet was only 22% of that observed when cells were incubated with a comparable amount of [methyl-3H]methionine. Both the binding of AdoMet and the methylation of phospholipids were inhibited by exogenous S-adenosyl-L-homocysteine. Therefore, the binding may be linked to a phospholipid methyltransferase.     

Photolabeling of CheR methyltransferase with S-adenosyl-L-methionine (AdoMet). Studies on the AdoMet binding site.

CheR methyltransferase from Salmonella typhimurium was directly photolabeled with S-adenosyl-L-[methyl-3H]methionine. The labeled protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was detected by fluorography. The methylase-S-adenosyl-L-methionine adduct was found to be stable under the experimental conditions employed. Labeling was found to be a function of the concentration of enzyme, S-adenosyl-L-methionine (AdoMet), and the intensity and time of UV irradiation. The extent of labeling and protein methylation was found to be inhibited by S-adenosyl-L-homocysteine, S-adenosyl-L-ethionine, and sinefungin, which are known to compete with AdoMet for the same binding site on the enzyme. Our earlier data showed that the enzyme has 2 cysteine residues and that these are important for enzyme activity. Here, we show that sulfhydryl reagents inhibit the photolabeling of the substrate to the enzyme, indicating the presence of cysteine in the vicinity of the substrate-binding site. We also found that when Cys31 was modified to Ser, no photolabeling of CheR was observed, whereas a modification of Cys229 to Ser had little effect on the ability of AdoMet to label the enzyme. This suggests that Cys31 is located at or near AdoMet-binding site. The labeled protein was cleaved at tryptophan residues, generating two major fragments, each containing 1 cysteine residue. SDS-PAGE and fluorography of the cleaved products indicated the presence of the label being associated with the Cys31 fragment. Similar results were obtained when the labeled protein was cleaved at glutamic acid residues using V8 protease. A tryptic digest of the labeled protein showed two radioactive peptide peaks when subjected to separation on reverse phase high pressure liquid chromatography. The labeled peptides were further digested to free amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that Cys31 may be involved with substrate binding, as well as with catalysis.     

Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases

An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. As an example, we have studied methylation of DNA by the EcoRV DNA methyltransferase. The reaction progress curves measured with this assay are linear with respect to time. Methylation rates linearly increase with enzyme concentration. The rates are comparable to results obtained with the same enzyme using a different assay. The biotin-avidin assay is inexpensive, convenient, quantitative, fast and well suited to process many samples in parallel. The accuracy of the assay is high, allowing to reproduce results within +/- 10%. The assay is very sensitive as demonstrated by the detection of incorporation of 0.8 fmol methyl groups into the DNA. Under the experimental conditions, this corresponds to methylation of only 0.03% of all target sites of the substrate. Using this assay, the DNA methylation activity of some M.EcoRV variants could be detected that was not visible by other in vitro methylation assays.     

Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase

A covalent complex between tRNA (m5U54)methyltransferase, 5-fluorouridine tRNA(Phe), and S-adenosyl-L-[methyl-3H]methionine was formed in vitro and purified. Previously, it was shown that in this complex the 6-position of fluorouridine-54 is covalently linked to a catalytic nucleophile and the 5-position is bound to the transferred methyl group of AdoMet [Santi, D. V., & Hardy, L. W. (1987) Biochemistry 26, 8599-8606]. Proteolysis of the complex generated a [3H]methyl-FUtRNA-bound peptide, which was purified by 7 M urea-15% polyacrylamide gel electrophoresis. The peptide component of the complex was sequenced by gas-phase Edman degradation and found to contain two cysteines. The tritium was shown to be associated with Cys 324 of the methyltransferase, which unequivocally identifies this residue as the catalytic nucleophile.     

Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates

Radioactivity from 3H-[methyl]-S-adenosyl-L-methionine (AdoMet) was covalently bound to protein-O-carboxylmethyltransferase and phenylethanolamine N-methyltransferase following 10-15 min irradiation by short-wave ultraviolet light. This photoaffinity binding of 3H-[methyl]-AdoMet was blocked by S-adenosylhomocysteine and sinefungin, but was not affected by 5 mM dithiothreitol. The binding was also inhibited by including methyl acceptors such as calmodulin (protein-O-carboxylmethyltransferase) or phenylethanolamine (phenylethanolamine N-methyltransferase) in the photoaffinity incubation. Staphlococcus V8 protease digests of 3H-[methyl]-AdoMet/enzyme complexes revealed that the primary structure around the AdoMet binding site is different in these two enzymes. Thus, protein-O-carboxylmethyltransferase, a large molecule methyltransferase, can covalently bind 3H-[methyl]-AdoMet in a manner similar to that of phenylethanolamine-N-methyltransferase.     

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Specificity of S-adenosyl-L-methionine in the inactivation and the labeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC, is irreversibly inactivated by its substrate AdoMet. AdoMet has two diastereomers with respect to its sulfonium center, (-)-AdoMet and (+)-AdoMet. We prepared (+)- and (-)-AdoMet from a commercial source, and compared their activities as a substrate and as an inactivator of ACC synthase isolated from tomato (Lycopersicon esculentum Mill). fruits. Only (-)-AdoMet produced ACC, whereas both (-)- and (+)-AdoMet inactivated ACC synthase; (+)-AdoMet inactivated the enzyme three times faster than (-)-AdoMet. We have previously shown that ACC synthase was specifically radiolabeled when the enzyme was incubated with S-adenosyl-L-[3,4-14C]methionine. The present results further indicate that S-adenosyl-L-[carboxyl-14C]methionine, but not S-adenosyl-L-[methyl-14C]methionine, radiolabeled the enzyme. These data suggest that the 2-aminobutyric acid portion of AdoMet is linked to ACC synthase during the autoinactivation process. A possible mechanism for ACC synthase inactivation by AdoMet is discussed.     

Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine

Methylenetetrahydrofolate reductase commits tetrahydrofolate-bound one carbon units to use in the regeneration of the methyl group of adenosylmethionine (AdoMet) in eucaryotes and its activity is allosterically inhibited by AdoMet. Limited proteolysis and scanning transmission electron microscopy have been employed to show that the enzyme is a dimer of identical subunits and that each subunit is composed of spatially distinct domains with molecular masses of approximately 40 and 37 kDa (Matthews, R. G., Vanoni, M. A., Hainfeld, J. F., and Wall, J. (1984) J. Biol. Chem. 259, 11647-11650). We now report the use of the photoaffinity label 8-azido-S-adenosylmethionine (8-N3AdoMet) to locate the binding site for the allosteric inhibitor on the 37-kDa domain. In the absence of light, 8-N3AdoMet is itself an inhibitor of methylenetetrahydrofolate reductase activity, with a Ki value 4.8-fold higher than AdoMet, and like AdoMet it induces slow transitions between active and inactive forms. Photoaffinity labeling is dependent on irradiation with ultraviolet light and is prevented by AdoMet but not by ATP. Limited proteolysis of the photolabeled enzyme results in the formation of a labeled 37-kDa fragment which is further processed to a labeled 34-kDa fragment. On conversion of the 34-kDa fragment to a 31-kDa polypeptide, all label is lost, suggesting that the labeling is restricted to an approximately 3-kDa region near one end of the 37-kDa polypeptide. Limited proteolysis of the native enzyme, while completely desensitizing the enzyme to inhibition by AdoMet or 8-N3AdoMet, does not prevent subsequent photolabeling of the 37-kDa peptide fragment. This photolabeling does not occur in the presence of excess AdoMet. These latter experiments suggest that the desensitization of the enzyme eliminates the ability of allosteric effectors to stabilize an inactive form of the enzyme, but does not abolish specific binding of 8-N3AdoMet or AdoMet.     

Site-specificity of histone H1 methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.     

Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding.

DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.     

Incorporation of Intracisternally Administered l-[methyl-3H]Methionine and S-Adenosyl-l-[methyl-3H]-Methionine into Rat Brain Phospholipids

The incorporation of intracisternally injected L-[methyl-3H]methionine [( 3H]Met) or S-adenosyl-L-[methyl-3H]methionine (Ado[3H]Met) into rat brain AdoMet and phospholipid pools was examined. When [3H]Met was administered, both AdoMet and phospholipid pools were labeled. However, exogenously injected Ado[3H]Met did not serve as a substrate for phospholipid-N-methyltransferases. It was concluded that only Ado[3H]Met formed in situ was utilized to methylate phospholipids and that this process was initiated on the cytoplasmic side of the membrane. The apparent biological half-life in brainstem of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine formed from [3H]Met was 1.4 and 1.7 days, respectively. The half-life of phosphatidylcholine could not be determined due to interference from peripheral sources.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Identification of amino acid residues photolabeled with 2-azido[alpha-32P]adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase

When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido[alpha-32P]adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled [Dalbon, P., Boulay, F., & Vignais, P. V. (1985) FEBS Lett. 180, 212-218]. The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.     

A quantum mechanics/molecular mechanics study of the catalytic mechanism and product specificity of viral histone lysine methyltransferase

There are three reaction steps in the S-adenosylmethionine (AdoMet) methylation of lysine-NH2 catalyzed by a methyltransferase. They are (i) combination of enzyme.Lys-NH3+ with AdoMet, (ii) substrate ionization to provide enzyme.AdoMet.Lys-NH2, and (iii) methyl transfer providing enzyme.AdoHcy.Lys-N(Me)H2+ and the dissociation of AdoHcy. In this study of the viral histone methyltransferase (vSET), we find that substrate ionization of vSET.Lys27-NH3+, vSET.Lys27-N(Me)H2+, and vSET.Lys27-N(Me)2H+ takes place upon combination with AdoMet. The presence of a water channel allows dissociation of a proton to the solvent. There is no water channel in the absence of AdoMet. That the formation of a water channel is combined with AdoMet binding was first discovered in our investigation of Rubisco large subunit methyltransferase. Via a quantum mechanics/molecular mechanics (QM/MM) approach, the calculated free energy barrier (DeltaG++) of the first methyl transfer reaction catalyzed by vSET [Lys27-NH2 + AdoMet --> Lys27-N(Me)H2+ + AdoHcy] equals 22.5 +/- 4.3 kcal/mol, which is in excellent agreement with the free energy barrier (21.7 kcal/mol) calculated from the experimental rate constant (0.047 min-1). The calculated DeltaG++ of the second methyl transfer reaction [AdoMet + Lys27-N(Me)H --> AdoHcy + Lys27-N(Me)2H+] at the QM/MM level is 22.6 +/- 3.6 kcal/mol, which is in agreement with the value of 22.4 kcal/mol determined from the experimental rate constant (0.015 min-1). The third methylation [Lys27-N(Me)2 + AdoMet --> Lys27-N(Me)3+ + AdoHcy] is associated with a DeltaG++ of 23.1 +/- 4.0 kcal/mol, which is in agreement with the value of 23.0 kcal/mol determined from the experimental rate constant (0.005 min-1). Our computations establish that the first, second, and third methyl transfer steps catalyzed by vSET are linear SN2 reactions with the bond making being approximately 50% associative.     

5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase

5-Fluorodeoxycytidine (FdCyd) was incorporated into a synthetic DNA polymer containing the GCGC recognition sequence of HhaI methylase to give a polymer with about 80% FdCyd. In the absence of AdoMet, poly(FdC-dG) bound competitively with respect to poly(dG-dC) (Ki = 3 nM). In the presence of AdoMet, the analogue caused a time-dependent, first-order (k = 0.05 min-1) inactivation of the enzyme. There is an ordered mechanism of binding in which enzyme first binds to poly(FdC-dG), then binds to AdoMet, and subsequently forms stable, inactive complexes. The complexes did not dissociate over the course of 3 days and were stable to heat (95 degrees C) in the presence of 1% SDS. Gel filtration of a complex formed with HhaI methylase, poly(FdC-dG), and [methyl-3H] AdoMet gave a peak of radioactivity eluting near the void volume. Digestion of the DNA in the complex resulted in a reduction of the molecular weight to the size of the methylase, and the radioactivity in this peak was shown to be associated with protein. These data indicate that the complexes contain covalently bound HhaI methylase, poly(FdC-dG), and methyl groups and that 5-fluorodeoxycytidine is a mechanism-based inactivator of the methylase. By analogy with other pyrimidine-modifying enzymes and recent studies on the mechanism of HhaI methylase (Wu & Santi, 1987), these results suggest that an enzyme nucleophile attacks FdCyd residues at C-6, activating the 5-position for one-carbon transfer.(ABSTRACT TRUNCATED AT 250 WORDS)