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.     

Time dependence of the methylation of myelin basic protein from bovine brain; evidence for protein-methylarginine demethylation

In the presence of excess S-adenosylmethionine (AdoMet), the extent of methylation of myelin basic protein (MBP) by partially purified AdoMet:MBP methyltransferase is a non-linear function of time, reaching a limiting value as available MBP is depleted and then decreasing monotonically. This decrease is not caused by proteolytic cleavage of MBP nor by effects related to substrate or product instability under the incubation conditions and is not observed in heat-inactivated samples. S-Adenosylhomocysteine is not required for the demethylation to occur, and with purified enzyme, the decrease is not observed. The data strongly suggest that the decrease in methyl content represents an enzyme-catalyzed demethylation reaction. This would represent the first report of an enzyme which catalyzes protein-methylarginine demethylation.     

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.     

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

Metabolism of S-adenosyl-L-methionine in intact human erythrocytes

Freshly isolated human erythrocytes contain S-adenosyl-L-methionine (AdoMet) at a concentration of about 3.5 mumol/l cells. When such cells are incubated in a medium containing 30 microM L-methionine, 18 mM D-glucose and 118 mM sodium phosphate (pH 7.4), intracellular AdoMet levels continuously decrease to a value of about 0.1 microM after 24 h. This occurs in spite of the fact that the cellular concentrations of the substrates for the AdoMet synthetase reaction, ATP and L-methionine, remain relatively constant. In a search for incubation conditions that lead to stable levels of AdoMet in incubated cells, we have developed a sodium-Hepes-buffered medium which includes 1 mM adenine and a stoichiometric excess of MgCl2 over its ligand, phosphate. The inclusion of magnesium ion (and a reduction in phosphate) appears to increase intracellular free Mg2+, which is required for full activity of the erythrocyte AdoMet synthetase. Even in the presence of MgCl2, however, the AdoMet pool level can drop 4-6-fold within the first 2 h of incubation. We present evidence that suggests that this initial fall in the cellular AdoMet level may be due to the activation of AdoMet-dependent protein carboxyl methyltransferase, an enzyme which accounts for a large fraction of the total cellular AdoMet utilization. Adenine, or related compounds in the medium may prevent this activation, although the mechanism of this action is not clear at present.     

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.     

Kinetic and catalytic mechanism of HhaI methyltransferase

Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.     

Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase.

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.     

Kinetic mechanism of isoprenylated protein methyltransferase.

The kinetic mechanism of the rod outer segment (ROS) isoprenylated protein methyltransferase was investigated. This S-adenosyl-L-methionine (AdoMet)-linked enzyme transfers methyl groups to carboxyl-terminal isoprenylated cysteine residues of proteins, generating methyl esters. The enzyme also processes simple substrates such as N-acetyl-S-farnesyl-L-cysteine (L-AFC). Initial studies showed that a ping-pong Bi Bi mechanism could be eliminated. In a ping-pong Bi Bi mechanism plots of 1/v versus 1/[substrate A] at different fixed substrate B concentrations are expected to yield a family of parallel lines whose slopes equal Km/Vmax. In fact, converging curves were found, which suggested a sequential mechanism. Dead-end inhibitors were used in order to further investigate the kinetic mechanism. S-Farnesylthioacetic acid is shown to be a dead-end competitive inhibitor with respect to the prenylated substrate L-AFC. On the other hand, S-farnesylthioacetic acid proved to be uncompetitive with respect to AdoMet, suggesting an ordered mechanism with AdoMet binding first. Further evidence for this mechanism came from product inhibition studies using the methyl ester of L-AFC (L-AFCMe) and S-adenosyl-L-homocysteine (AdoHcy). Since AdoMet binds first to the enzyme, one of the products (L-AFCMe or AdoHcy) should be a competitive inhibitor with respect to it. It could be shown that AdoHcy is a competitive inhibitor with respect to AdoMet, but L-AFCMe is a mixed-type inhibitor both with respect to AdoMet and to L-AFC. Therefore, AdoHcy combines with the same enzyme form as does AdoMet, and must be released from the enzyme last. Moreover, L-AFC and L-AFCMe must bind to different forms of the enzyme.     

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.     

Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.     

The kinetic mechanism of S-adenosyl-L-methionine: glutamylmethyltransferase from Salmonella typhimurium

The kinetic mechanism of the CheR methyltransferase, S-adenosyl-L-methionine (AdoMet): protein-L-glutamate O-methyltransferase (EC 2.1.1.24), from Salmonella typhimurium was investigated. Initial velocity, product inhibition, and binding studies were performed, and from the data obtained, it was determined that the mechanism of the reaction catalyzed by the enzyme is random. Initial velocity rates were measured with varied amounts of both substrates, and double-reciprocal plots gave patterns which converged on or near the abscissa. The products, S-adenosyl-L-homocysteine and methylated receptor, were found to be competitive inhibitors with respect to both AdoMet and receptor. Equilibrium dialysis and immunoprecipitation studies indicated that the two substrates can bind to the enzyme independent of each other. These results are consistent with a random mechanism with no abortive complexes being formed. The Michaelis constants calculated for AdoMet and receptor were 8.62 microM and 2.03 mg/ml total membrane protein (approximately 2.10 microM Tar protein), and the apparent dissociation constants of AdoMet and the receptor were 16.8 microM and 4.07 mg/ml total membrane protein (approximately 4.2 microM Tar protein), respectively. The Kd of AdoMet for the enzyme was 10.9 microM as determined by binding studies.     

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 microM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 microM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.     

Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis

tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.     

Catalytic properties and kinetic mechanism of human recombinant Lys-9 histone H3 methyltransferase SUV39H1: participation of the chromodomain in enzymatic catalysis

Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation and chromatin organization. SUV39H1 methylates H3K9 at the pericentric heterochromatin region and participates in the maintenance of genome stability. In this study, a recombinant purified SUV39H1 is used for substrate specificity and steady-state kinetic analysis with peptides representing the un- or dimethylated lysine 9 histone H3 tail or full-length human recombinant H3 (rH3). Recombinant SUV39H1 methylated its substrate via a nonprocessive mechanism. Binding of either peptide or AdoMet first to the enzyme made a catalytically competent binary complex. Product inhibition studies with SUV39H1 showed that S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of AdoMet, suggesting a random mechanism in a bi-bi reaction for recombinant SUV39H1 in which either substrate can bind to the enzyme first and either product can release first. The turnover numbers (k(cat)) for the H3 tail peptide and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower compared to that of the unmethylated peptide. The Michaelis constants for AdoMet (K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and rH3, respectively. A reduction in the level of methylation was observed at high concentrations of rH3, implying substrate inhibition. Deletion of the chromodomain or point mutation of the conserved amino acids, W64A or W67A, of SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for catalysis.     

Slow binding inhibition of S-adenosylmethionine synthetase by imidophosphate analogues of an intermediate and product.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.     

Reconciling structure and function in HhaI DNA cytosine-C-5 methyltransferase

Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme.DNA complex and the lack of catalytic competence of the enzyme.S-adenosyl-L-methionine (AdoMet) complex. The enzyme.AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme.AdoMet.DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme.AdoMet and ternary enzyme. S-adenosyl-L-homocysteine.DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA. We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the 5-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi- and unmethylated substrates alter the fates and lifetimes of discrete enzyme.substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.     

Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase.

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.