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Kinetic Mechanism of Cytosine DNA MethyltransferaseMspI

1999; Elsevier BV; Volume: 274; Issue: 21 Linguagem: Inglês

10.1074/jbc.274.21.14743

1083-351X

Sanjoy K. Bhattacharya, Ashok K. Dubey,

RNA modifications and cancer

A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of λ-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (k cat/K M) of 0.9 × 108m−1s−1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated λ-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). TheS-adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per mol of enzyme s−1) which is similar to catalytic constants (k cat = ∼0.056 s−1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first. A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of λ-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (k cat/K M) of 0.9 × 108m−1s−1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated λ-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). TheS-adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per mol of enzyme s−1) which is similar to catalytic constants (k cat = ∼0.056 s−1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first. A class of DNA methyltransferases, referred to as m5C-DNA MTase, 1The abbreviations used are: Mtase, methyltransferase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; bp, base pair; meDNA, methylated DNA. catalyzes transfer of a methyl group from the physiological methyl donor,S-adenosyl-l-methionine (AdoMet), to C-5 of cytosine in the substrate DNA in a sequence-specific manner. In case of procaryotes, it primarily forms part of the restriction-modification system. But in higher organisms, it has been implicated to have a role in a variety of cellular functions such as developmental process (1Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar), transposition (2Fedoroff N.V. Cell. 1989; 77: 473-476Google Scholar), recombination (3Engler P. Weng A. Storb U. Mol. Cell. Biol. 1993; 13: 571-577Crossref PubMed Scopus (66) Google Scholar), X-chromosome inactivation (4Gartler S.N. Riggs A.D. Annu. Rev. Genet. 1983; 17: 155-190Crossref PubMed Scopus (478) Google Scholar), and genomic imprinting (5Li E. Beard C. Jaenisch R. Nature. 1993; 266: 362-365Crossref Scopus (1784) Google Scholar). All the m5C-DNA MTases, whether from virus, bacteria, or higher organisms, share a common architectural plan (6Posfai J. Bhagwat A.S. Posfai G. Roberts R.J. Nucleic Acids Res. 1989; 17: 2421-2435Crossref PubMed Scopus (437) Google Scholar). They have six highly conserved and four not so well conserved motifs (7Kumar S. Chang X. Klimasauskas S. Mi S. Posfai J. Roberts R.J. Wilson G.G. Nucleic Acids Res. 1994; 22: 1-10Crossref PubMed Scopus (394) Google Scholar). Although both adenine and cytosine DNA MTases show a degree of similarity in their architectural plan, there are important differences (8Timinskas A. Butkus V. Janulaitis A. Gene (Amst.). 1995; 157: 3-11Crossref PubMed Scopus (86) Google Scholar). Conservation of sequence is more prominent among members of the m5C-DNA MTase family than among those belonging to theN 6-adenine DNA MTase family (8Timinskas A. Butkus V. Janulaitis A. Gene (Amst.). 1995; 157: 3-11Crossref PubMed Scopus (86) Google Scholar). Kinetics of methyl transfer by M.HhaI (m5C-MTase) and M.EcoRI (N 6-adenine MTase) has been elucidated (9Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar, 10Reich N.O. Mashhoon N. Biochemistry. 1991; 30: 2933-2939Crossref PubMed Scopus (98) Google Scholar). Despite both being bilobal and having a degree of similarity in structure, there are marked differences in the kinetic mechanism of these two methyltransferases. Both are known to be consistent with an ordered Bi Bi steady state mechanism. However, whereas the M.HhaI binds DNA first, the M.EcoRI binds AdoMet first (9Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar, 10Reich N.O. Mashhoon N. Biochemistry. 1991; 30: 2933-2939Crossref PubMed Scopus (98) Google Scholar). Although the catalytic mechanism for m5C-DNA MTases is well understood, significant differences do exist among them from a kinetic standpoint. Two of the m5C-MTases, M.Dcm and M.BspRI, transfer the methyl group to themselves when incubated with AdoMet in the absence of DNA (11Hanck T. Schmidt S. Fritz H.-J. Nucleic Acids Res. 1993; 21: 303-309Crossref PubMed Scopus (44) Google Scholar, 12Szilak L. Finta C. Patthy A. Venetianer P. Kiss A. Nucleic Acids Res. 1994; 22: 2876-2881Crossref PubMed Scopus (8) Google Scholar). This unique feature of suicidal self-methylation has not been reported for any other enzyme of the m5C-DNA MTase family. The M.MspI displays many important features, which are uncommon to other m5C-MTases. For instance, it has the largest N-terminal sequence (107 bases) among the methylases of bacterial restriction/modification systems (7Kumar S. Chang X. Klimasauskas S. Mi S. Posfai J. Roberts R.J. Wilson G.G. Nucleic Acids Res. 1994; 22: 1-10Crossref PubMed Scopus (394) Google Scholar), it induces a bend in the substrate DNA in a sequence-specific manner (13Dubey A.K. Bhattacharya S.K. Nucleic Acids Res. 1997; 25: 2025-2029Crossref PubMed Scopus (11) Google Scholar), and it has also been shown to possess a topoisomerase activity (14Matsuo K. Silke J. Gramatikoff K. Schaffner W. Nucleic Acids Res. 1994; 22: 5354-5359Crossref PubMed Scopus (38) Google Scholar). In view of these unique features of the M.MspI and the fact that it recognizes DNA substrates differently than its isoschizomer M.HpaII, we attempted to investigate its kinetic mechanism. In the present communication, we report the kinetic parameters for methylation and tritium exchange reactions of the M.MspI, and a kinetic analysis of product inhibition and presteady state kinetics. Escherichia coli K-12 strain ER 1727 [_(mcr BC−) hsdRMS− mrr)2::Tn 10,mcrA1272:: Tn 10, F′lacproABlacIq_(lacZ)−M15] was used for overexpression of the target gene which was placed downstream of the T7 promoter regulated by the lac operator in the expression vector pMSP (15Mi S. Roberts R.J. Nucleic Acids Res. 1992; 20: 4811-4816Crossref PubMed Scopus (51) Google Scholar). The E. coli strain ER 1727 harboring recombinant plasmid pMSP was cultivated in Luria Broth containing 150 μg ml−1 ampicillin. The cells were induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside and harvested as reported previously (13Dubey A.K. Bhattacharya S.K. Nucleic Acids Res. 1997; 25: 2025-2029Crossref PubMed Scopus (11) Google Scholar). The harvested cells (0.5 g) were suspended in 2 ml of buffer containing 10 mm potassium phosphate (pH 7.4), 1 mm EDTA, 14 mm β-mercaptoethanol, 0.3m NaCl, and 10% glycerol. The cell suspension was sonicated for 3 min with burst and gap periods of 15 s at 14-μm amplitude in a Sonirep 150 (New Brunswick, Edison, NJ). The temperature during sonication was maintained at 5 ± 2 °C. The sonicated cell suspension was centrifuged at 31,000 × g for 30 min. The cell-free extract, so recovered, was treated as a crude preparation from the MspI methylase. The enzyme was purified to apparent homogeneity on the following ion-exchange columns: phosphocellulose, S-Sepharose, and Q-Sepharose as described previously (16Dubey A.K. Mollet B. Roberts R.J. Nucleic Acids Res. 1992; 20: 1579-1585Crossref PubMed Scopus (33) Google Scholar). Active fractions were pooled and dialyzed against the buffer containing 50 mm Tris-HCl, pH 8.0, 0.1 m NaCl, 10 mm EDTA, 1 mm dithiothreitol, and 50% glycerol for storage and further use. To determine the temperature where the M.MspI displayed optimum activity, methylation reactions were carried out at different temperatures in the range of 10–40 °C in NS buffer (50 mm Tris-HCl, pH 7.5; 10 mm EDTA; 1 mm β-mercaptoethanol with 200 μg/ml bovine serum albumin). The reaction mixtures were preincubated for 20 min at the corresponding temperature in a heat block (VWR Scientific) prior to enzyme addition. For estimation of the optimum value of pH for the M.MspI activity, assay buffers with pH values in the range of 6.5–9.0 were used, and the methylase activity was determined at the corresponding reaction pH. Quantitative assay of the methyltransferase activity was performed by measuring the incorporation of [3H]methyl group into the substrate DNA according to the procedures described elsewhere (9Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar, 16Dubey A.K. Mollet B. Roberts R.J. Nucleic Acids Res. 1992; 20: 1579-1585Crossref PubMed Scopus (33) Google Scholar) with a few modifications. Briefly, the following procedures were adopted for quantitative methyltransferase assay. For procedure a, the methylation reaction involving 50 nm32P-DNA (1459-bp BstXI fragment from φX174 DNA) and 200 nm [3H]AdoMet (15 Ci/mmol) was carried out in a buffer containing 50 mm Tris-HCl, pH 7.5, 10 mm EDTA, 1 mm dithiothreitol, and 10% glycerol at 37 °C; 10-μl aliquots from a 30-μl reaction mixture were withdrawn at intervals of 1 min for a total period of 30 min and added to a 300-μl slurry of DE52 (50% v/v in H2O). Prior to addition of the reaction mixture, a 1 mm solution of AdoHcy was mixed with the slurry in 1:1 ratio to quench the MTase activity. The resin was washed with six 1-ml portions of 0.2m NH4HCO3, followed each time by a 2-min centrifugation at 1000 rpm. After a final wash with 1 ml of H2O, the adsorbed radioactivity was eluted from the resin by addition of 600 μl of acidified milli-Q water (550 μl of H2O + 50 μl of 1 n HCl). The supernatant was recovered following centrifugation at 1000 rpm, and radioactivities (3H and 32P) were counted. Determination of concentrations of 32P-DNA and [3H] methyl group was based on a correlation between the disintegrations/min and the molar concentration. For 32P-DNA this correlation was established by determining the DNA concentration spectrophotometrically and obtaining the disintegrations/min value from the scintillation counter. For [3H]AdoMet, the values of disintegrations/min were from the known concentration of [3H]AdoMet as provided by the supplier. According to these determinations 2200 dpm corresponded to 1 pmol of double-stranded DNA and 33,300 dpm of methyl- 3H corresponded to 1 pmol of AdoMet. For procedure b, another set of reactions for the assay of the M.MspI activity involved the use of unlabeled 1459-bpBstXI fragment of φX174 as substrate under the typical reaction conditions described above. The reactions were initiated by adding serially diluted M.MspI solutions (3 μl), and the reaction was incubated for 1 min. A 20-μl sample from a 30-μl reaction mixture was spotted on DE81 filter discs placed in a filter manifold. The filter discs were washed twice with 0.2 mNH4HCO3, twice with 80% EtOH in 50 mm phosphate buffer (pH 8.0), and once with 90% EtOH in 50 mm phosphate buffer (pH 8.0). The filters were dried under vacuum suction on the filter manifold and subsequently under a lamp prior to measurement of the radioactivity in 4 ml of scintillant (Supertron, Kontron). The amount of enzyme that transferred 1 pmol of [3H]methyl group to the substrate DNA was thus obtained. One unit of the M.MspI activity was defined as the amount of enzyme that incorporated 1 pmol of [3H]methyl group into the DNA at saturating concentration of the substrates (50 nm DNA, ∼200 nm AdoMet) in 1 min at 37 °C. The specific activity of M.MspI referred to units/mg protein, that is picomoles of [3H]methyl group/min/mg of protein. The procedure for determination of protein concentration was based on Bradford's principle (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) and was performed by using the Bio-Rad Coomassie plus kit. Standard curves were established using bovine serum albumin. Electrophoretic analyses of protein samples were carried out in 10% SDS-polyacrylamide gels using Laemlli buffer (18Zwieb C. Kim J. Adhaya S. Jost J.P. Saluz H.P. A Laboratory Guide to in Vitro Studies of Protein-DNA Interactions. Birkhauser Verlag, Berlin1991: 245-257Crossref Google Scholar). Proteins were visualized by staining with Coomassie Brilliant Blue. SalI, EcoRI, andSalI-HindIII fragments (121 bp) containing a single M.MspI-specific site (5′-CCGG-3′), prepared from plasmid pBend2 (18Zwieb C. Kim J. Adhaya S. Jost J.P. Saluz H.P. A Laboratory Guide to in Vitro Studies of Protein-DNA Interactions. Birkhauser Verlag, Berlin1991: 245-257Crossref Google Scholar), were used as DNA substrates. A third DNA substrate was prepared using XhoI digestion of pBend2 to produce a linear 121-bp fragment containing the 5′-CCGG-3′ sequence 19 bp from the 5′ end. This third substrate was different than the first two with regard to the position of the 5′-CCGG-3′ sequence along the molecule. Although the first two substrates had identical length as well as position of the 5′-CCGG-3′ along the molecule (70 bp from the 5′ end), they differed from one another with respect to bases flanking the canonical sequence. A larger substrate of 1459 bp was derived from φX174 DNA using BanI digestion (the 5′-CCGG-3′ sequence was 84 bp from the 5′ end). This substrate was cyclized (13Dubey A.K. Bhattacharya S.K. Nucleic Acids Res. 1997; 25: 2025-2029Crossref PubMed Scopus (11) Google Scholar) and digested with BstXI to obtain the same length DNA fragment with the 5′-CCGG-3′ position being 702 bp from the 5′ end. The hemi-methylated DNA was generated as follows. A synthetic oligonucleotide, 5′-CCTAG-3′, was ligated to one strand of the double-stranded 1459-bp BanI fragment to create size difference between the strands. It was subsequently methylated using excess of AdoMet and M.MspI. The strands of the modified as well as the unmodified 1459-bp fragments were separated on 5% polyacrylamide gel electrophoresis containing 6 m urea. The complementary strands were mixed in equimolar ratio to obtain hemi-methylated DNA. All the DNA substrates were purified from native polyacrylamide gel electrophoresis by electroelution in a dialysis bag (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and reprecipitated. Their concentrations were determined spectrophotometrically (18Zwieb C. Kim J. Adhaya S. Jost J.P. Saluz H.P. A Laboratory Guide to in Vitro Studies of Protein-DNA Interactions. Birkhauser Verlag, Berlin1991: 245-257Crossref Google Scholar) using aliquots after redissolving the precipitated DNA. The 1:1 annealing of the hemi-methylated substrate was confirmed by studying thermal denaturation/renaturation kinetics by determining the absorbance at 260 nm spectrophotometrically. Features of the various DNA substrates, obtained as above, are summarized in Table I.Table IFeatures of different fragments of DNA used as substrate for M.MspI in kinetic analysisOrigin/sourceSizeFragmentPosition of CCGG from the 5′ endbase pairspBend2121XhoI19pBend2121SalI–EcoRI70pBend2121SalI–HindIII70φX1741459BanI84φX1741459BstXI702φX1741459:1464; hemi-methylatedBstXI702In addition to the above defined substrate containing a single M.MspI-specific methylation site, 50-kilobase pair λ-DNA molecule was used as a much longer substrate for the enzyme with 328 sites per molecule. Both intact and the sonicated forms of the λ-DNA were used in evaluation of the kinetic parameters. Open table in a new tab In addition to the above defined substrate containing a single M.MspI-specific methylation site, 50-kilobase pair λ-DNA molecule was used as a much longer substrate for the enzyme with 328 sites per molecule. Both intact and the sonicated forms of the λ-DNA were used in evaluation of the kinetic parameters. The data from kinetic studies were analyzed as described by Wu and Santi (9Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) using the FORTRAN program of Cleland (20Cleland W.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1967; 29: 1-32PubMed Google Scholar, 21Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1929) Google Scholar). The statistical criteria of Cleland (21Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1929) Google Scholar) were used in the current analysis. Kinetic studies were done using all of the DNA substrates that were prepared from plasmid pBend2 and φX174. The determination of initial velocity was made by performing the methyltransferase assay under conditions described earlier. In a series of identical reactions containing 0.03 nm M.MspI (specific activity ∼145 × 108 units mg−1) and 200 nm [3H]AdoMet, the concentration of the substrate DNA was varied in the range of 1–25 nm. A double-reciprocal plot of the initial velocity versus DNA concentration allowed the determination ofK MDNA and V max. Similarly, initial velocities were obtained by varying the concentration of [3H]AdoMet in the range of 5–50 nm while keeping the DNA concentration fixed at 50 nm and keeping other reaction conditions identical. The double-reciprocal plot of initial velocity versus[3H]AdoMet concentration allowed the determination ofK MAdoMet andV max. The catalytic constant (k cat) was calculated as the ratio ofV max (0.1 nm min−1) to the enzyme concentration used (0.03 nm), taking 49 kDa as the molecular mass of the M.MspI. The kinetic constants were also determined for intact and sonicated λ-DNA. While determining initial velocities, the product formation was measured under such conditions that overall inhibition of the reaction by AdoHcy (generated) was less than 5%. Thus the highest AdoHcy/AdoMet ratio allowed in the present experiments was ∼0.03. The 1459-bp BstXI fragment was used as the DNA substrate for the experiments pertaining to product inhibition kinetics. The methylated DNA (meDNA) fragments used for substrate inhibition studies were obtained by incubating 600 nm DNA with 1.2 mm[3H]AdoMet in a reaction volume of 30 μl for a period of 100 min using 0.03 nm enzyme. After being extracted twice with chloroform it was precipitated with ethanol, dried, and used. The product inhibition studies were performed under identical conditions as described for the quantitative MTase assay. Inhibition by AdoHcy was studied using 200 nm [3H]AdoMet (fixed concentration) while keeping the AdoHcy concentrations fixed (0.0, 2.0, 4.0, and 6.0 nm) and varying the concentration of substrate DNA from 1 to 25 nm for each of the fixed concentrations of AdoHcy. Similarly, another series of identical reactions included 50 nm substrate DNA (fixed concentration); AdoHcy concentrations fixed at 0.0, 1.0, 2.0, 3.0 nm and [3H]AdoMet concentrations varied in the range of 5–50 nm for each of the fixed concentrations of AdoHcy. The double-reciprocal plots of the initial velocityversus DNA/[3H]AdoMet concentrations were obtained at each concentration of the AdoHcy. These plots were used to determine the values of K iAdoHcy for DNA and for [3H]AdoMet by following the procedure described elsewhere (22Hiromi K. Kinetics of Fast Enzyme Reactions: Theory and Practice. John Wiley and Sons Inc., New York1979Google Scholar). For inhibition by meDNA, the AdoMet concentration was kept constant at 200 nm and that of the DNA was varied in the range of 1–20 nm against each of the chosen concentrations of the meDNA as follows: 0.0, 2.5, 5.0, and 7.7 nm. Similarly, another series of identical reactions was carried out at the fixed concentration of DNA (50 nm) and different concentrations of [3H]AdoMet ranging from 5 to 100 nm against each of the fixed concentrations of the meDNA as follows: 0.0, 3.0, and 7.0 nm. The data from these experiments were used to generate double-reciprocal plots of initial velocityversus variable substrate to determine the values ofK imeDNA for DNA and for AdoMet. In a 50-μl reaction volume of 5 μg of 1459-bp BstXI, DNA was labeled with 10 μl of [γ-32P]ATP (5 × 106cpm/μmol, NEN Life Science Products) and 40 units of T4 polynucleotide kinase (New England Biolabs) for 1 h at 37 °C. The DNA was purified by passing through (twice) a NAP-5 column (Amersham Pharmacia Biotech). The labeled DNA was precipitated and resuspended in 10 μl of TE buffer. In a 20-μl volume, 2 μm M.MspI was preincubated with 2 μm radiolabeled DNA (1459-bp BstXI DNA) for 2 min at 37 °C. For reaction initiation, a 4-μl aliquot of this mixture was removed and brought to a final volume of 200 μl with MTase buffer that contained 2 μm labeled DNA and 0.5 μm [3H]AdoMet. This mixture (after addition of a 4-μl aliquot as above) was incubated for a total period of 30 s; 20-μl aliquots were withdrawn at intervals of 5 s for activity measurements. Another reaction was set up where 4-μl aliquots of preincubation mix of M.MspI (2 μm)-labeled DNA (2 μm) as above were made up to 200 μl with MTase buffer that contained 2 μmunlabeled DNA and 0.5 μm [3H]AdoMet. This mixture was incubated for 30 s as before, and 20-μl samples were withdrawn at 5-s intervals and analyzed as above. The moles of productmeDNA per mol of enzyme were calculated for all of these reactions. These data were used to obtain a plot of mole ofmeDNA per mol of enzyme versus time for further analysis. About 300 nm[methyl- 3H]DNA (1459-bp BstXI fragment) was incubated with 0.03 nm MspI methylase in the presence of 400 nm AdoHcy for a period of 30–100 min at 37 °C in a reaction volume of 30 μl prepared in the assay buffer described earlier. A 20-μl sample was withdrawn and analyzed for the MTase activity by the quantitative method (preparation b) mentioned above. The samples were spotted on DE81 filter discs and were counted for radioactivity using a scintillation counter to determine loss of radioactivity from the [methyl- 3H]DNA. A tritiated DNA was prepared by employing the forward reaction of the enzyme wherein 600 nm DNA (1459-bp BstXI fragment) was incubated in tritiated water (specific activity >6 × 105 dpm/mmol) with the M.MspI (1.5 nm) for about 100 min. The reaction was terminated by heating the reaction mixture at 70 °C for 5 min. The DNA was recovered by precipitation with absolute alcohol. The precipitation step was repeated twice; the precipitate was washed with 70% alcohol each time. The DNA preparations were digested with micrococcal nuclease (Amersham Pharmacia Biotech) and with calf spleen phosphodiesterase (Roche Molecular Biochemicals, Germany). The digestion products were subsequently analyzed on thin layer chromatography plates, which were phosphorimaged in a FUJI BAS2000 PhosphorImager using tritium-sensitive TR FUJI BAS2000 plates, to confirm the tritium incorporation into the cytosine. Release of tritium from [3H]DNA fragment, prepared above, was measured by a charcoal-binding assay. The exchange reaction was performed in a 30-μl reaction volume containing 0.5–2.5 nm [3H]DNA (specific activity 3 × 105 dpm/mmol) and 0.03 nm MspI methylase that was incubated at 37 °C for 30 min. Samples (10 μl) were withdrawn at an interval of 1 min for analysis. These samples were pipetted into a 0.99-ml suspension of Norit (charcoal, 7.5% w/v in 0.12 n HCl). The charcoal suspension was mixed with 490 μl of MilliQ water and centrifuged (12,000 × g, 2 min), and the resulting supernatant (500 μl) was recovered and filtered through glass wool in a double Eppendorf assembly, the upper one having a hole with glass wool. The filtrate (500 μl) so recovered was added to 4 ml of Supertron Scintillant (Kontron) for radioactivity measurement in a scintillation counter. The velocity of the M.MspI-catalyzed release of tritium from [3H]DNA per min was calculated. The effect of AdoHcy (0.0, 2.5, 5.0, 7.5, and 10 nm) onV max of tritium release was determined using an identical assay. Furthermore, the effect of AdoMet at a level of 10 nm on the M.MspI-catalyzed tritium exchange was also observed. The M.MspI preparations purified to apparent homogeneity, as judged by SDS-polyacrylamide gel electrophoresis, were used for the kinetic analysis. These preparations displayed optimal activity at a pH value of 7.5 and at the temperature of 36 ± 1 °C. An activation energy of 15.7 kJ mol was obtained for the enzyme from the Arrhenius plot (ln k/T versus 1/T; data not shown). The kinetics of methyl transfer on the C-5 of the target base (outer cytosine in the 5′-CCGG-3′ sequence) by the M.MspI has been investigated using a variety of substrates, which differed from one another with respect to (i) length in base pairs, (ii) position of the target sequence from the left end in the linear molecule, (iii) bases flanking the canonical sequence, and (iv) the methylation state of the strands. These variations in substrates allowed an examination of the kinetic parameters as affected by them and would further be helpful in elucidating the kinetic mechanism for the M.MspI. The progress of the M.MspI-catalyzed methylation of the 1459-bp fragment was determined in order to evaluate the period during which the rate of product formation remains linear. The progress curve was obtained by measuring the amount of product formed at different time intervals. A plot of the methylated DNA product versus time was obtained. The M.MspI-catalyzed methylation of the 1459-bp fragment by AdoMet progressively decreases as the reaction proceeds (Fig. 1A). The inhibition appears to be competitive with respect to AdoMet, and the nonlinear kinetics is consistent with competition by the AdoHcy generated in the reaction. I-catalyzed methylation reaction fits Equation 1, P/t=Vmax1−(KM/Ki)+KM(Ki+So)·1/tKM−Kiln SoSo−PEquation 1 which describes a reaction wherein the product, AdoHcy, shows competitive inhibition with respect to the substrate, AdoMet (23Orsi B.A. Tipton K.F. Methods Enzymol. 1979; 63: 159-183Crossref PubMed Scopus (152) Google Scholar).P is the amount of AdoHcy formed at time t, So is the initial AdoMet concentration, andK i is the dissociation constant of AdoHcy. The amount of AdoHcy formed corresponds to the amount of methylated DNA, the product which is measured in these experiments. Plots ofP/t versus (ln(So/(So −P))/t provide a series of lines with positive slopes (Fig. 1B) indicating that the K Mof AdoMet is much larger than K i. The kinetic parameters (K MDNA,K MAdoMet, andV max) were estimated from the double-reciprocal plots of initial velocity versus substrate concentration. The catalytic constant (k cat) was calculated as the ratio of initial velocity to that of protein concentration. The specificity constant was obtained ask cat/K M values. The catalytic constant and turnover number for the M.MspI are synonymous as it is envisaged to have only one catalytic site per protein molecule. Values for all the kinetic parameters for different DNA substrates and AdoMet have been listed in TableII.Table IIKinetic parameters of M.MspI-catalyzed methylation of DNADNA substrates; size in bp (fragment)KMDNAKMAdoMetkcatVmaxkcat/KMnmnms −1nmmin −1m −1 s −1 (×10 6)121 bp (XhoI)7.69 ± 0.5116.1 ± 0.440.0560.1 ± 0.017.28121 bp (SalI–EcoRI)7.14 ± 0.4316.32 ± 0.380.0560.1 ± 0.0057.8121 bp (SalI–HindIII)7.14 ± 0.2216.32 ± 0.610.0560.1 ± 0.0067.81459 bp (BanI)4.34 ± 0.180.0560.1 ± 0.00712.91459 bp (BstXI)2.28 ± 0.0313.5 ± 0.540.0560.1 ± 0.006241459:1464 bp (BstXI) (hemi-methylated DNA)1.85 ± 0.050.0560.1 ± 0.00530λ-DNA, 50 kbakb, kilobase pair. (328-M.MspI sites)1.8 ± 0.0212.7 ± 0.330.170.3 ± 0.00890λ-DNA (sonicated)4.4 ± 0.3113.7 ± 0.430.0560.1 ± 0.00512.7a kb, kilobase pair. Open table in a new tab These experiments were performed to determine the effect of different concentrations of DNA and AdoMet on the initial velocity. The initial velocity data also allowed a determination of the substrate inhibition of Ad