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• W1973496342 abstract "We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction fromS-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNAMe andS-adenosyl-l-homocysteine, AdoHcy) release was AdoMet↓DNA↓DNAMe↑AdoHcy↑. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNAMe dissociates relatively rapidly (koff = 1.7 s−1) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (koff = 0.018 s−1), indicating that its release is the rate-limiting step, consistent withkcat = 0.015 s−1. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations. We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction fromS-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNAMe andS-adenosyl-l-homocysteine, AdoHcy) release was AdoMet↓DNA↓DNAMe↑AdoHcy↑. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNAMe dissociates relatively rapidly (koff = 1.7 s−1) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (koff = 0.018 s−1), indicating that its release is the rate-limiting step, consistent withkcat = 0.015 s−1. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations. methyltransferase S-adenosyl-l-methionine S-adenosyl-l-homocysteine methylated DNA 5-hydroxymethylcytosine-containing T4 αgt− βgt−(unglucosylated) DNA model selection criterion DNA methyltransferases (MTases)1 are involved in a variety of important cellular functions in both prokaryotes and eukaryotes (1Ahmad I. Rao D.N. Crit. Rev. Biochem. Mol. Biol. 1996; 31: 361-380Crossref PubMed Scopus (67) Google Scholar, 2Dryden D.T. Cheng X. Blumenthal R.M. S-Adenosylmethionine-dependent Methyltransferases: Structures and Functions. World Scientific, Singapore1999: 283-340Crossref Google Scholar, 3Vertino P.M. Cheng X. Blumenthal R.M. S-Adenosylmethionine-dependent Methyltransferases: Structures and Functions. World Scientific, Singapore1999: 341-372Crossref Google Scholar). In addition, DNA MTases are excellent subjects for detailed studies of specific protein-DNA interaction because they are highly specific and have a comparatively simple organization. Elucidating the kinetic mechanism of the reactions catalyzed by these enzymes still remains an important problem to investigate in the area of biological DNA methylation. Kinetic schemes have been proposed forHhaI (4Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar, 5O'Gara M. Zhang X. Roberts R.J. Cheng X. J. Mol. Biol. 1999; 287: 201-209Crossref PubMed Scopus (65) Google Scholar, 6Lindstrom W.M. Flynn J. Reich N.O. J. Biol. Chem. 2000; 275: 4912-4919Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Vilkaitis G. Merkiene E. Serva S. Weinhold E. Klimasauskas S. J. Biol. Chem. 2001; 276: 20924-20934Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), MspI (8Bhattacharya S.K. Dubey A.K. J. Biol. Chem. 1999; 274: 14743-14749Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), human Dnmt1 (9Bacolla A. Pradhan S. Roberts R.J. Wells R.D. J. Biol. Chem. 1999; 274: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and murine Dnmt1 (10Flynn J. Reich N.O. Biochemistry. 1998; 37: 15162-15169Crossref PubMed Scopus (53) Google Scholar) [C5-cytosine]MTases, and forEcoRI (11Reich N.O. Mashhoon N. Biochemistry. 1991; 30: 2933-2939Crossref PubMed Scopus (98) Google Scholar, 12Reich N.O. Mashhoon N. J. Biol. Chem. 1993; 268: 9191-9193Abstract Full Text PDF PubMed Google Scholar), EcaI (13Szilak L. Der A. Deak F. Venetianer P. Eur. J. Biochem. 1993; 218: 727-733Crossref PubMed Scopus (27) Google Scholar), TaqI (14Wolcke J. The kinetic mechanism of the DNA methyltransferase from Thermus aquaticus and selection of a DNA-binding peptide by means of phage displayPh. D. Dissertation. University Dortmund, Germany1998Google Scholar),EcoRV (15Gowher H. Jeltsch A. J. Mol. Biol. 2000; 303: 93-110Crossref PubMed Scopus (68) Google Scholar), and Type III EcoP15I [N6-adenine]MTases (16Rao D.N. Page M.G. Bickle T.A. J. Mol. Biol. 1989; 209: 599-606Crossref PubMed Scopus (41) Google Scholar). All of these enzymes exhibit a sequential bi-bi mechanism; however, they differ with respect to the order of substrate binding and rate-limiting step. For instance, whereas the rate of methyl group transfer is at least 300-fold faster than the rate of dissociation of the products for EcoRI (12Reich N.O. Mashhoon N. J. Biol. Chem. 1993; 268: 9191-9193Abstract Full Text PDF PubMed Google Scholar), in contrast, the rate of methyl group transfer is the rate-limiting step in the TaqI methylation reaction (14Wolcke J. The kinetic mechanism of the DNA methyltransferase from Thermus aquaticus and selection of a DNA-binding peptide by means of phage displayPh. D. Dissertation. University Dortmund, Germany1998Google Scholar). Furthermore, several different modes of MTase binding to the substrates DNA (17Klimasauskas S. Szyperski T. Serva S. Wuthrich K. EMBO J. 1998; 17: 317-324Crossref PubMed Scopus (104) Google Scholar, 18Allan B.W. Reich N.O. Beechem J.M. Biochemistry. 1999; 38: 5308-5314Crossref PubMed Scopus (80) Google Scholar) and AdoMet or its analogs (5O'Gara M. Zhang X. Roberts R.J. Cheng X. J. Mol. Biol. 1999; 287: 201-209Crossref PubMed Scopus (65) Google Scholar, 19Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (108) Google Scholar) have been distinguished. In addition, changes in enzyme conformation (isomerization) associated with binding substrate DNA and/or AdoMet may also influence the reaction kinetics. Allosteric activation by methylated DNA was observed with human Dnmt1 MTase (9Bacolla A. Pradhan S. Roberts R.J. Wells R.D. J. Biol. Chem. 1999; 274: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (199) Google Scholar). The binding of two AdoMet molecules was reported for the EcoDam (21Bergerat A. Guschlbauer W. Nucleic Acids Res. 1990; 18: 4369-4375Crossref PubMed Scopus (72) Google Scholar) and PvuII MTases (22Adams G.M. Blumenthal R.M. Biochemistry. 1997; 36: 8284-8292Crossref PubMed Scopus (31) Google Scholar, 23Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (166) Google Scholar), and an effector role was suggested for the second AdoMet molecule. The precise binding stoichiometry of AdoMet, as well as its double role as methyl donor and allosteric effector, remains to be fully characterized for T4 Dam (24Kossykh V.G. Schlagman S.L. Hattman S.M. Nucleic Acids Res. 1993; 21: 4659-4662Crossref PubMed Scopus (22) Google Scholar, 25Malygin E.G. Evdokimov A.A. Zinoviev V.V. Ovechkina L.G. Lindstrom W.M. Reich N.O. Schlagman S.L. Hattman S.M. Nucleic Acids Res. 2001; 29: 2361-2369Crossref PubMed Google Scholar). Thus, in contrast to the apparently universal ternary structure of the catalytic domains (23Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (166) Google Scholar,26Schluckebier G. O'Gara M. Saenger W. Cheng X. J. Mol. Biol. 1995; 247: 16-20Crossref PubMed Scopus (236) Google Scholar, 27O'Gara M. McCloy K. Malone T. Cheng X. Gene. 1995; 157: 135-138Crossref PubMed Scopus (24) Google Scholar, 28Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (193) Google Scholar), the existing biochemical data are complex and not clear enough for generalizations to be made concerning the kinetics of the reactions catalyzed by the different DNA MTases. Therefore, further investigation is required. The Dam DNA-[N6-adenine]MTase encoded by phage T4 catalyzes methyl group transfer from AdoMet to the N6position of an Ade residue in the palindromic sequence, 5′-GATC-3′ (30Schlagman S.L. Hattman S. Gene. 1983; 22: 139-156Crossref PubMed Scopus (40) Google Scholar). T4 Dam belongs to the large family of α-group type II DNA MTases (31Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (429) Google Scholar), in which there are at least 50 members known today and almost half of them are [N6-adenine] GATC-specific isoschizomers. Furthermore, the enzymes that modify the N6-amino nitrogen of adenine share not only the nine conserved motifs, but also possess striking similarity in their target recognition domains, although they have different targets;e.g. GATC, GANTC, GATATC, and non-palindromic CATCC (28Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Such homology may, in turn, indicate that a considerable amount of similarity exists in their three-dimensional structures, as well as in their mechanism of action. Recently, more progress has been made in the study of this family of enzymes; the first crystal structure for the GATC-specific DpnM MTase complexed with AdoMet was reported (28Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and an extensive biochemical investigation of the GATATC-specificEcoRV MTase was performed (15Gowher H. Jeltsch A. J. Mol. Biol. 2000; 303: 93-110Crossref PubMed Scopus (68) Google Scholar, 32Cal S. Connolly B.A. J. Biol. Chem. 1997; 272: 490-496Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 33Roth M. Helm-Kruse S. Friedrich T. Jeltsch A. J. Biol. Chem. 1998; 273: 17333-17342Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 34Jeltsch A. Roth M. Friedrich T. J. Mol. Biol. 1999; 285: 1121-1130Crossref PubMed Scopus (34) Google Scholar, 35Roth M. Jeltsch A. Nucleic Acids Res. 2001; 29: 3137-3144Crossref PubMed Scopus (19) Google Scholar). Previously, catalytic turnover and Michaelian constants were obtained for the T4 Dam methylation reaction using either polymeric 5-hydroxymethylcytosine-containing T4 αgt−βgt− (unglucosylated)dam− (unmethylated) DNA (hmCyt-DNA) or a set of synthetic oligonucleotide duplexes containing one or more defect(s) or substitution(s) in the target sequence (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 37Zinoviev V.V. Evdokimov A.A. Gorbunov Y.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar, 38Malygin E.G. Zinoviev V.V. Petrov N.A. Evdokimov A.A. Jen-Jacobson L. Kossykh V.G. Hattman S. Nucleic Acids Res. 1999; 27: 1135-1144Crossref PubMed Scopus (30) Google Scholar). In this communication, we report a detailed steady state kinetic analysis of the methylation of a synthetic 20-mer oligonucleotide duplex catalyzed by T4 Dam; and, we propose a kinetic reaction scheme for this member of the α-group GATC-family of DNA MTases. [3H-CH3]S-adenosyl-l-methionine (15 Ci/mmol; 1 mCi/ml) was purchased from Amersham Biosciences.S-adenosyl-l-homocysteine (AdoHcy) and sinefungin were from Sigma. Unlabeled AdoMet (Sigma) was purified further by chromatography on a C18 reversed-phase column as described previously (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The synthetic 20-mer duplex used as substrate had the following sequence shown in Structure I.5′­CAGTTTAGGATC¯CATTTCAC­3′3′­GTCAAATCCTAG¯GTAAAGTG­5′STRUCTURE IA modified duplex containing N6-methyladenine in the recognition sequence GATC (underlined) in both strands was used in the product inhibition studies. Oligonucleotides were synthesized on an Applied Biosystems 380A/380B DNA synthesizer, and concentrations were determined spectrophotometrically. The duplexes were obtained by heating and annealing complementary oligonucleotide chains from 90 to 20 °C over 7–12 h. T4 Dam was purified to homogeneity as described previously (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The protein concentration was determined by the Bradford method (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar), which yielded values in close agreement with those determined spectrophotometrically at 280 nm from the known composition and molar extinction coefficients of individual aromatic amino acid residues in pH 6.5, 6.0 m guanidinium hydrochloride, 0.02 m phosphate buffer (40Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5048) Google Scholar). DNA MTase assays were similar to that previously reported (38Malygin E.G. Zinoviev V.V. Petrov N.A. Evdokimov A.A. Jen-Jacobson L. Kossykh V.G. Hattman S. Nucleic Acids Res. 1999; 27: 1135-1144Crossref PubMed Scopus (30) Google Scholar). T4 Dam reactions were carried out at 25 °C in buffer containing 100 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 5% glycerol, and 0.2 mg/ml bovine serum albumin (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). A low concentration of T4 Dam (1 nm) was used for most experiments to increase the accuracy of the calculation of the initial reaction velocities by reducing the influence of any burst in product formation (37Zinoviev V.V. Evdokimov A.A. Gorbunov Y.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar). The concentrations of AdoMet, substrate DNA, and inhibitors varied according to the individual experiment. The reactions were initiated by addition of prewarmed T4 Dam to preincubated mixtures of [3H-CH3]AdoMet and substrate DNA (with or without inhibitors; final volume is 25 μl). The reaction time used was selected to ensure initial velocity conditions; i.e.product formation was less than 15% of the initial substrate and added product inhibitor concentrations during the time of the reaction. At appropriate intervals, an aliquot (15 μl) was withdrawn from each mixture and spotted on a DE81 anion-exchange filter disc (Whatman, 1.5 cm). Filters were washed three times with 0.02 mNH4HCO3, twice with water, once with 75% ethanol, and then dried. They were counted in a toluene liquid scintillator. The molar concentrations of [3H-CH3] groups incorporated into DNA were quantified as described previously (41Thielking V. Dubois S. Eritja R. Guschlbauer W. Biol. Chem. 1997; 378: 407-415PubMed Google Scholar). The validity of the quantification procedure was confirmed under complete methylation conditions (about 1 h at a 1:2 enzyme/substrate ratio), where the calculated concentrations of [3H-CH3] groups incorporated into DNA coincided with the reaction mixture concentrations of methylatable Ade residues. All experiments were done at least twice. Kinetic data were analyzed using the program Scientist 2.01 (MicroMath®) for non-linear regression analysis. We used the statistical model selection criterion (MSC) recommended by program developers to determine the goodness of fit for each kinetic model. The MSC is defined by the formula in Equation1, MSC=ln∑i=1nwi(Yobsi−Ȳobs)2∑i=1nwi(Yobsi−Ycali)2−2pnEquation 1 where n is the number of points,wi are the weights applied to each point,Yobs is the weighted mean of the observed data and p is the number of parameters. The model that has the largest MSC value is by definition the best or most appropriate model. The ability of T4 Dam to catalyze the transfer of a methyl group from methylated DNA to AdoHcy was tested using an [N6-3H-methyl]adenine-containing 20-mer DNA duplex at a concentration of 200 nm. Reactions were performed in the presence of 50 nm T4 Dam and 0, 5, or 50 μm AdoHcy. No tritium loss from the [N6-3H-methyl]adenine containing 20-mer was observed over a period of 270 min (data not shown). This indicated that the reverse reaction is at least 500-fold slower than the forward one. Therefore, the transfer of the methyl group from AdoMet to DNA can be considered irreversible for the T4 Dam MTase; this has been previously shown for the HhaI (4Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) andMspI (8Bhattacharya S.K. Dubey A.K. J. Biol. Chem. 1999; 274: 14743-14749Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) MTases. Initial velocities (V) were determined at various concentrations of the substrates, [3H-CH3]AdoMet and 20-mer duplex DNA. The substrate concentrations used in these experiments were up to 5-fold above Km for AdoMet (Km AdoMet = 490 nm) and 15-fold above Km for DNA (Km DNA = 6.3 nm) (37Zinoviev V.V. Evdokimov A.A. Gorbunov Y.A. Malygin E.G. Kossykh V.G. Hattman S. Biol. Chem. 1998; 379: 481-488Crossref PubMed Scopus (35) Google Scholar). As shown in Fig. 1, both double-reciprocal plots gave a series of straight lines that intersected to the left of the 1/V axis, which rules out a ping-pong bi-bi mechanism. An ordered rapid-equilibrium mechanism is also unlikely because the double-reciprocal plot lines should have intersected at the 1/V axis for the second substrate that binds. In addition, secondary plots of the slopes and 1/V intercepts versus reciprocal concentrations of substrates were approximately linear (not shown), permitting calculation of conventional kinetic parameters. In accordance with graphical predictions, the experimental data fit an equation that corresponds to either a steady state-ordered bi-bi or a rapid-equilibrium random bi-bi mechanism (42Cleland W.W. Biochim. Biophys. Acta. 1963; 67: 104-137Crossref PubMed Google Scholar) according to Equation2. V=Vmax·[A]·[B]/(KiA·KmB+KmA·[B]+KmB·[A]+[A]·[B])Equation 2 Because the conversion step (kmeth = 0.56 s−1) in the T4 Dam methylation reaction (43Malygin E.G. Lindstrom W.M. Schlagman S.L. Hattman S.M. Reich N.O. Nucleic Acids Res. 2000; 28: 4207-4211Crossref PubMed Scopus (27) Google Scholar) was much faster than the catalytic turnover constant (kcat = 0.015 s−1), a rapid equilibrium random mechanism appears to be ruled out. Results of product inhibition studies below support this conclusion. Product inhibition studies are commonly performed to determine whether there is a preferential order of substrate binding for a particular multiple substrate reaction (44Rudolph F.B. Methods Enzymol. 1979; 63: 411-436Crossref PubMed Scopus (90) Google Scholar). We used this approach to determine whether T4 Dam first binds substrate DNA or AdoMet (Fig.2 and TableI). We found that AdoHcy was a competitive inhibitor with respect to AdoMet (Fig. 2A) and a non-competitive inhibitor with respect to 20-mer DNA duplex (Fig.2B); this is consistent with previous results (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Similar inhibition patterns were also obtained with sinefungin, another non-reactive AdoMet analog (data not shown). The other reaction product, fully methylated 20-mer DNA duplex, exhibited non-competitive inhibition with respect to both AdoMet and unmethylated 20-mer DNA duplex (Fig. 2, C and D). Secondary plots of the slopes and 1/V intercepts versus concentration of inhibitor were approximately linear (not shown). Thus, these product inhibition patterns (Table I) are consistent with a steady state-ordered bi-bi mechanism (44Rudolph F.B. Methods Enzymol. 1979; 63: 411-436Crossref PubMed Scopus (90) Google Scholar), in which the substrate binding and product release order are AdoMet↓DNA↓DNAMe↑AdoHcy↑.Table IProduct inhibition analysis of reaction catalyzed by the T4 Dam MTaseInhibitorVariable substrateFixed substrateInhibition typeAdoHcyAdoMetDNACompetitiveSinefunginAdoMetDNACompetitiveAdoHcyDNAAdoMetNoncompetitiveSinefunginDNAAdoMetNoncompetitiveDNAMeAdoMetDNANoncompetitiveDNAMeDNAAdoMetNoncompetitive Open table in a new tab Generally, DNA MTases may form binary complexes with either of their reaction substrates. Binding of AdoMet in the absence of substrate DNA has been confirmed, in particular by co-crystal structures (5O'Gara M. Zhang X. Roberts R.J. Cheng X. J. Mol. Biol. 1999; 287: 201-209Crossref PubMed Scopus (65) Google Scholar, 19Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (108) Google Scholar, 23Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (166) Google Scholar, 28Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and by copurification of MTase with tightly bound AdoMet (45Piekarowicz A. Brzezinski R. J. Mol. Biol. 1980; 144: 415-429Crossref PubMed Scopus (25) Google Scholar, 46Kumar S. Cheng X. Pflugrath J.W. Roberts R.J. Biochemistry. 1992; 31: 8648-8653Crossref PubMed Scopus (60) Google Scholar, 47Friedrich T. Roth M. Helm-Kruse S. Jeltsch A. Biol. Chem. 1998; 379: 475-480Crossref PubMed Scopus (15) Google Scholar). On the other hand, MTases are capable of recognizing and binding at specific DNA sequence(s) and flipping the target base in the absence of AdoMet or its non-reactive analogs (17Klimasauskas S. Szyperski T. Serva S. Wuthrich K. EMBO J. 1998; 17: 317-324Crossref PubMed Scopus (104) Google Scholar, 34Jeltsch A. Roth M. Friedrich T. J. Mol. Biol. 1999; 285: 1121-1130Crossref PubMed Scopus (34) Google Scholar, 48Reinisch K.M. Chen L. Verdine G.L. Lipscomb W.N. Cell. 1995; 82: 143-153Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 49Holz B. Klimasauskas S. Serva S. Weinhold E. Nucleic Acids Res. 1998; 26: 1076-1083Crossref PubMed Scopus (197) Google Scholar); although, as a rule, AdoMet (or its non-reactive analogs) increases the enzyme's affinity for substrate DNA (13Szilak L. Der A. Deak F. Venetianer P. Eur. J. Biochem. 1993; 218: 727-733Crossref PubMed Scopus (27) Google Scholar, 17Klimasauskas S. Szyperski T. Serva S. Wuthrich K. EMBO J. 1998; 17: 317-324Crossref PubMed Scopus (104) Google Scholar, 21Bergerat A. Guschlbauer W. Nucleic Acids Res. 1990; 18: 4369-4375Crossref PubMed Scopus (72) Google Scholar, 50Szczelkun M.D. Connolly B.A. Biochemistry. 1995; 34: 10724-10733Crossref PubMed Scopus (63) Google Scholar, 51Powell L.M. Murray N.E. Nucleic Acids Res. 1995; 23: 967-974Crossref PubMed Scopus (27) Google Scholar, 52Malygin E.G. Petrov N.A. Gorbunov Y.A. Kossykh V.G. Hattman S.M. Nucleic Acids Res. 1997; 25: 4393-4399Crossref PubMed Scopus (34) Google Scholar). However, DNA MTases can also form stable, non-functional (dead-end) enzyme-product-substrate ternary complexes, as has been observed for the MTase-AdoHcy-DNA complex ofHhaI (53O'Gara M. Klimasauskas S. Roberts R.J. Cheng X. J. Mol. Biol. 1996; 261: 634-645Crossref PubMed Scopus (160) Google Scholar). Thus, if the initial concentration of the second substrate to bind were sufficiently high, a steady state-ordered reaction (which has a specific order of substrate binding and product release) would show inhibition of the initial reaction velocity due to the formation of non-productive binary and/or dead-end ternary complexes (54Cleland W.W. Methods Enzymol. 1979; 63: 500-513Crossref PubMed Scopus (148) Google Scholar). In contrast, a rapid equilibrium random bi-bi mechanism should show no substrate inhibition when the initial concentration of either (or both) substrate(s) is high (54Cleland W.W. Methods Enzymol. 1979; 63: 500-513Crossref PubMed Scopus (148) Google Scholar). The results in Fig.3 show a strong inhibition in T4 Dam initial reaction velocity at high concentrations of substrate 20-mer DNA duplex. This indicates that T4 Dam does not obey a rapid equilibrium random bi-bi mechanism. Rather, the data are consistent with a steady state-ordered bi-bi mechanism, as we had concluded from the product inhibition analysis. In contrast to the results with the substrate 20-mer DNA duplex, increasing the concentration of AdoMet led to a progressive stimulation in the reaction rate (Fig. 4). Whereas the initial portion of the concentration dependence curve corresponded approximately to a conventional hyperbolic dependence (Fig. 4,inset), saturation was never achieved. In fact, the rate of the reaction was linearly proportional to the AdoMet concentration up to 30 μm (60-fold above the Km for AdoMet). We will present possible explanations of this unexpected effect in the “Discussion.” The HhaI (4Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) and MspI (8Bhattacharya S.K. Dubey A.K. J. Biol. Chem. 1999; 274: 14743-14749Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) DNA MTases have been analyzed for their ability to perform the reverse of the methylation reaction; e.g. transferring the methyl group from [methyl-3H]DNA to AdoHcy to form [methyl-3H]AdoMet. Both enzymes were incapable of performing the reverse catalytic reaction, and we have shown here that T4 Dam is also unable to do so. Therefore, the T4 Dam-catalyzed methylation reaction appears to be irreversible, which has facilitated further kinetic analysis. The product inhibition patterns obtained for the T4 Dam MTase (Fig. 2, Table I) are consistent with a steady state-ordered bi-bi mechanism, in which the order of substrate binding and product release are AdoMet↓DNA↓DNAMe↑AdoHcy↑. However, the stimulatory effect of high AdoMet concentrations on DNA methylation rate indicates that the mechanism is more complex; that is, the release of AdoHcy from the MTase-AdoHcy complex appears to be associated with the binding of AdoMet in a concerted event (discussed below). In a previous study, polymeric T4 αgt−βgt− (unglucosylated)dam+ hmCyt-DNA (previously methylated in vitro by T4 Dam) was observed to be a competitive inhibitor of unmethylated substrate hmCyt-DNA (36Kossykh V.G. Schlagman S.L. Hattman S.M. J. Biol. Chem. 1995; 270: 14389-14393Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In the experiments reported here; however, methylated 20-mer DNA duplex was a non-competitive inhibitor of the unmethylated 20-mer DNA duplex DNA. The difference in the nature of the inhibition can be explained, as a formal analysis indicates (not shown here), by a preferentially processive methylation mechanism of polymeric DNA, in contrast to a distributive methylation mechanism of the 20-mer duplex. This question is currently under investigation. The preferential order of substrate binding proposed for T4 Dam is identical to that found for two other [N6-adenine]MTases, EcoRI (11Reich N.O. Mashhoon N. Biochemistry. 1991; 30: 2933-2939Crossref PubMed Scopus (98) Google Scholar" @default.
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• W1973496342 date "2002-01-01" @default.
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• W1973496342 title "Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase" @default.
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