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INTRODUCTION |
A class of DNA methyltransferases, referred to as m5C-DNA
MTase,1 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 (1),
transposition (2), recombination (3), X-chromosome inactivation (4),
and genomic imprinting (5). All the m5C-DNA MTases, whether from virus,
bacteria, or higher organisms, share a common architectural plan (6).
They have six highly conserved and four not so well conserved motifs
(7). Although both adenine and cytosine DNA MTases show a degree of
similarity in their architectural plan, there are important differences
(8). Conservation of sequence is more prominent among members of the
m5C-DNA MTase family than among those belonging to the
N6-adenine DNA MTase family (8). Kinetics of
methyl transfer by M.HhaI (m5C-MTase) and M.EcoRI
(N6-adenine MTase) has been elucidated (9, 10).
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 (9, 10). 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 (11, 12). 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 (7), it induces a bend in the substrate DNA in a
sequence-specific manner (13), and it has also been shown to possess a
topoisomerase activity (14). 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.
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EXPERIMENTAL PROCEDURES |
Bacterial Strain and Plasmid
Escherichia coli K-12 strain ER 1727 [_(mcr BC
) hsd
RMS
mrr)2::Tn 10, mcrA1272:: Tn 10, F'lacproAB
lacIq_(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 (15). 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 mM
isopropyl-1-thio-
-D-galactopyranoside and harvested as
reported previously (13).
Purification of MspI Methylase
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
-mercaptoethanol, 0.3 M 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 (16). 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.
Optimum Reaction Conditions and Assays for the M.MspI
Activity
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
-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 (9, 16)
with a few modifications. Briefly, the following procedures were
adopted for quantitative methyltransferase assay.
For procedure a, the methylation reaction involving 50 nM
32P-DNA (1459-bp BstXI fragment from
X174
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 0.2 M 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-bp
BstXI fragment of
X174 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 M
NH4HCO3, 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.
Protein Analysis
The procedure for determination of protein concentration was
based on Bradford's principle (17) 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 (18).
Proteins were visualized by staining with Coomassie Brilliant Blue.
Determination of Kinetic Parameters
Substrates for Determination of Kinetic
Parameters--
SalI, EcoRI, and
SalI-HindIII fragments (121 bp) containing a
single M.MspI-specific site (5'-CCGG-3'), prepared from
plasmid pBend2 (18), 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
X174 DNA using BanI digestion (the 5'-CCGG-3' sequence
was 84 bp from the 5' end). This substrate was cyclized (13) 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
(19) and reprecipitated. Their concentrations were determined
spectrophotometrically (18) 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.
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Table I
Features of different fragments of DNA used as substrate for M.MspI in
kinetic analysis
In addition to the above defined substrate containing a single
M.MspI-specific methylation site, 50-kilobase pair -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 -DNA
were used in evaluation of the kinetic parameters.
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Kinetics of DNA Methylation Catalyzed by MspI Methylase--
The
data from kinetic studies were analyzed as described by Wu and Santi
(9) using the FORTRAN program of Cleland (20, 21). The statistical
criteria of Cleland (21) were used in the current analysis.
Kinetic studies were done using all of the DNA substrates that were
prepared from plasmid pBend2 and
X174. 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 of KMDNA and Vmax.
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 of
KMAdoMet and
Vmax. The catalytic constant
(kcat) was calculated as the ratio of
Vmax (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
-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.
Product Inhibition Kinetics--
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 velocity
versus DNA/[3H]AdoMet concentrations were
obtained at each concentration of the AdoHcy. These plots were used to
determine the values of Ki AdoHcy for
DNA and for [3H]AdoMet by following the procedure
described elsewhere (22). 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 velocity versus variable substrate to determine the values of
KimeDNA for DNA and for AdoMet.
Presteady State Isotope Partition Analysis--
In a 50-µl
reaction volume of 5 µg of 1459-bp BstXI, DNA was labeled
with 10 µl of [
-32P]ATP (5 × 106
cpm/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 µM
unlabeled 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 product
meDNA per mol of enzyme were calculated for all of these
reactions. These data were used to obtain a plot of mole of
meDNA per mol of enzyme versus time for further analysis.
Reverse Methylation and Catalysis of Tritium Exchange by
M.MspI--
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) on
Vmax 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.
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RESULTS |
Kinetics of DNA Methylation Catalyzed by the MspI
Methyltransferase--
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.
Progress Curve for the Methylation Reaction and Its
Analysis--
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.

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Fig. 1.
Progress curve analysis of the methylation
reaction of MspI DNA methyltransferase. Time
course of methylation of reaction contained 2 µM 1459-bp
DNA, the indicated concentrations of [3H]AdoMet (15 µCi/mmol) (5, 10, 15, and 20 nM), and 0.03 nM
MspI methylase under standard reaction conditions. Samples
at 1 min intervals were withdrawn and analyzed as described under
"Experimental Procedures." The data plotted in A were
fitted in Equation 1 to obtain values that are contained in
B.
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The progress curves of the M.Msp--
I-catalyzed methylation
reaction fits Equation 1,
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(Eq. 1)
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which describes a reaction wherein the product, AdoHcy, shows
competitive inhibition with respect to the substrate, AdoMet (23).
P is the amount of AdoHcy formed at time t,
So is the initial AdoMet concentration, and
Ki 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 of
P/t versus (ln(So/(So
P))/t provide a series of lines with positive
slopes (Fig. 1B) indicating that the KM
of AdoMet is much larger than Ki.
Determination of Kinetic Parameters--
The kinetic parameters
(KMDNA,
KMAdoMet, and
Vmax) were estimated from the double-reciprocal
plots of initial velocity versus substrate concentration.
The catalytic constant (kcat) was calculated as
the ratio of initial velocity to that of protein concentration. The
specificity constant was obtained as
kcat/KM 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 Table II.
Dependence of Initial Velocity on DNA and AdoMet
Concentrations--
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 AdoMet on DNA and substrate inhibition of DNA
on AdoMet, respectively (Fig. 2,
A and B). Initial velocity data were determined
at various concentrations of [3H]AdoMet and DNA (1459-bp
BstXI fragment). The double-reciprocal plot of
1/V versus 1/DNA concentration (1459-bp
BstXI) gave a series of lines intersecting close to but not
on 1/V axis (Fig. 2A) and vice versa
(Fig. 2B). These data therefore establish that AdoMet exerts
noncompetitive substrate inhibition on DNA and vice versa.
These data, therefore, suggest that a Ping-Pong mechanism is unlikely.
All data were fit to PING-PONG, EQUARD and SEQUEN programs. However,
the data were best fit to SEQUEN program, consistent with ordered
steady state or random rapid equilibrium mechanisms.

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Fig. 2.
Kinetics of DNA methylation catalyzed by the
MspI methyltransferase. Reactions contained the
indicated concentrations of 1459-bp DNA and [3H]AdoMet
(15 µCi/mmol) in standard buffer at 37 °C. M.MspI
was added to a final concentration of 0.03 nM. The samples
were withdrawn at 1-min intervals and assayed as described under
"Experimental Procedures." A, double-reciprocal plots of
the rates of methylation versus DNA (1459 bp) concentration
at different fixed concentration of AdoMet. B, reciprocal
plots of the rates of methylation versus AdoMet
concentration at different fixed concentration of DNA.
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Product Inhibition Kinetics--
Product inhibition analyses were
used to distinguish between the ordered and the random mechanisms. The
double-reciprocal plots of initial velocity versus
concentrations of DNA with respect to different fixed concentrations of
methylated DNA yielded a series of lines intersecting at the
y axis (Fig. 3A).
The methylated DNA inhibition is, thus, competitive with respect to
varying DNA as substrate. The methylated DNA inhibition with respect to
varying AdoMet yielded a series of lines that intersect to the left of the y axis, indicating inhibition to be noncompetitive (Fig.
3B). The AdoHcy inhibition with respect to varying AdoMet
concentration yielded a series of lines intersecting at the
y axis (Fig. 3C). The AdoHcy inhibition is
therefore competitive with respect to AdoMet. The AdoHcy inhibition
with respect to varying DNA concentration yielded parallel lines (Fig.
3D) and is therefore uncompetitive with respect to DNA. The
inhibition constant was obtained by equating the intersection point to
(1 + [I]/Ki/Vmax), where
[I] is concentration of the inhibitor. In the case of the
uncompetitive inhibition (Fig. 3D), each point intersecting
the initial velocity axis was equated to the above relationship to
obtain the inhibition constant. The product inhibition analysis has
been summarized in Table III.

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Fig. 3.
Product inhibition analysis of methylation
reaction catalyzed by M.MspI reactions contained the
indicated concentrations of 1459-bp DNA and [3H]AdoMet
(15 µCi/mmol) in standard buffer at
37 °C. M.MspI was added to a final concentration of
0.03 nM. The samples were withdrawn and assayed as
described under "Experimental Procedures." A,
double-reciprocal plots of the rates of methylation versus
1459-bp DNA concentrations at different fixed concentration of
methylated DNA. B, reciprocal plots of rates of methylation
versus AdoMet concentration at different fixed concentration
of methylated DNA. C, reciprocal plots of the rates of
methylation versus AdoMet concentrations at different fixed
concentration of AdoHcy. D, double-reciprocal plots of rates
of methylation versus 1459-bp DNA concentration at different
fixed concentration of AdoHcy.
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Presteady State Isotope Partitioning Studies--
Presteady state
kinetic analysis was used to elucidate which steps limit the catalytic
turnover. Preincubation of M.MspI with 2 µM
labeled DNA results in a "burst" of product formation upon reaction
initiation with addition of AdoMet
[methyl-3H]AdoMet and labeled DNA (Fig.
4, line A). The burst is
followed by a constant rate of product formation (0.06 mol of
meDNA per mol of enzyme s
1) similar to
catalytic turnover constant under steady state conditions (Fig. 4,
line A). A decreased burst size was observed when
unlabeled DNA and AdoMet [methyl-3H] AdoMet
was substituted in the chase. This burst is, however, much smaller in
size than that observed in A (Fig. 4, line
B). The data indicate that the M.MspI-DNA complex
is kinetically competent and preclude any kinetic mechanism that
requires AdoMet to be bound first. The methyl transfer, as evident from
presteady state analysis, precedes rate-limiting step(s). The DNA
methylation is followed by a slower step (or steps), and the slower
step is detected as the catalytic rate constant.

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Fig. 4.
Isotope partitioning analysis of
MspI DNA methyltransferase. Presteady state
kinetics of the MspI DNA methyltransferase, starting with
the MTase-DNA complex. After a 4-µl preincubation of MTase (2 µM) and labeled DNA (2 µM), AdoMet (0.5 µM) was diluted 50-fold into labeled DNA (2 µM) and AdoMet (0.5 µM) (line
A) or unlabeled DNA (2 µM) and AdoMet (0.5 µM) (line B).
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Reverse Reaction and Catalysis of Tritium Exchange by the MspI
Methylase--
The reverse reaction analysis was made to determine if
the M.MspI catalyzes transfer of a methyl group from the
[methyl-3H]DNA to AdoHcy. Within the
sensitivity limits of our detection, we could not detect any transfer
of a methyl from DNA to AdoHcy over a period of 100 min. Therefore, as
far as our present investigation is concerned, the
M.MspI-catalyzed reaction is irreversible under the
experimental conditions described here.
The MspI methylase-catalyzed exchange of tritium from C-5 of
the target cytosine with the hydrogen of water has been examined in the
absence of AdoMet. Tritium exchange from the [3H]DNA
substrates by the M.MspI was recorded in these experiments. The kinetic parameters for the M.MspI-catalyzed tritium
exchange were obtained in a manner identical to that for methylation
and are listed in Table IV. Evidently,
the rate of tritium exchange is ~3.3 times faster compared with the
rate of target base methylation. The
(kcat/KM) value of 7.4 × 107 M
1 s
1
represents a minimum value for the association of DNA (Fig.
5 and Table IV). This tritium exchange
demonstrates that the MspI MTase is capable of interacting
with DNA in the absence of AdoMet. However, AdoMet when present,
inhibits the tritium exchange (Fig. 6A). Furthermore, the velocity
of tritium exchange is affected by AdoHcy (Fig. 5 and Fig.
6B).

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Fig. 5.
Double-reciprocal plots of the rates of
5-3H exchange versus concentration of the
substrate DNA (1459-bp BstXI fragment) at different
fixed concentration of AdoHcy. Reaction (30 µl) contained
1459-bp DNA, 0.03 nM MspI, and indicated amounts
of AdoHcy. After incubation, samples were withdrawn and analyzed as
described under "Experimental Procedures."
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Fig. 6.
M.MspI-catalyzed
release of tritium from [3H]DNA substrate.
A, two identical reactions containing 2.5 nM
1459-bp DNA and 0.03 nM MspI methyltransferase
in standard buffer at 37 °C. One of the reactions contained 10 nM AdoMet; the other did not have added AdoMet. At the
indicated times, duplicate 10-µl samples were removed from each
reaction and measured for 3H2O by charcoal
assay as described under "Experimental Procedures." B,
inhibition of 5-3H exchange from 1459-bp DNA by AdoHcy
reactions (30 µl) contained 2.5 nM 1459-bp DNA, 0.03 nM MspI methylase, and the indicated
concentration of AdoHcy was used. After a 1-min interval 10 µl of
duplicate samples were withdrawn and assayed as described under
"Experimental Procedures."
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DISCUSSION |
Substrate Variability and the Kinetic Properties of the MspI
Methylase--
The M.MspI prefers the long chain substrates
over the short chain ones, as revealed by the relative affinities in
terms of the values of KM DNA for the DNA substrates
of varying length: 121- and 1459-bp and the 50-kilobase pair
-DNA
(Table II). The relative preferences for a long chain over short chain molecules of DNA as substrates have been observed with other type II
MTases as well. For example, the EcoRI MTase has a
KMDNA of 0.346 nM for the
linear pBR322 compared with 2.44 nM for a 14-mer
oligonucleotide (10), and the BamHI MTase has
KMDNA values of 3.2 and 11.2 nM with NdeI-linearized and
EcoRI-linearized molecules of pBR322, respectively (24). The
position of the target base along the linear DNA molecule also appears
to affect the affinity of the M.MspI for the substrate. It
was more evident with the 1459-bp substrate where the affinity for the
centrally located target (BstXI fragment;
KMDNA 2.28 ± 0.03 nM)
was nearly twice compared with that when it was located toward the end
(BanI fragment; KMDNA
4.34 ± 0.18 nM). For the EcoRI methylase,
an increase in the values of
kcat/KM with increase in the
length of DNA or with increase in the distance from the site to the
nearest end was observed earlier (10, 25). Whereas length of the
substrate DNA and the position of the recognition sequence along the
molecule had significant affect on the MTase affinity, the bases
flanking the canonical sequence did not apparently contribute toward
enzyme affinity for the substrate since similar values for the kinetic constants were recorded for the SalI-EcoRI and
the SalI-HindIII fragments (121 bp) in the
M.MspI-catalyzed reaction (Table II). Furthermore, the
methylation status of the recognition sequence had a profound effect on
the kinetic parameters as well since a hemi-methylated 1459-bp fragment
proved a better substrate for the enzyme. The higher affinity (1.85 nM) for hemi-methylated substrates, reported here, is in
agreement with the previous observations (26, 27). This increased
affinity for the hemi-methylated DNA, however, does not lead to an
enhanced rate of methyl transfer. The methylation reaction of the
M.MspI with intact
-DNA yielded a catalytic constant of
0.17 s
1 which was higher compared with the value of 0.056 s
1 recorded with sonicated
-DNA (Table II). The
kinetic parameters obtained with the sonicated
-DNA is comparable to
those obtained with 1459-bp fragment. The kinetic behavior of the
M.MspI with the intact and the sonicated
-DNA suggested a
processive mode of action for this enzyme rather than a distributive
mode. This is in agreement with a previous report that, based on
similar experiments, concluded that cytosine methylase isolated from
rat liver possessed a processive mechanism (28).
Kinetic Mechanism for the MspI Methylase--
The large
specificity constants (of the order of 107) for the
M.MspI-catalyzed methylation are inconsistent with any
random mechanism (where specificity constants are of the order of
103) and suggest an ordered steady state mechanism (22).
The methylated DNA in the M.MspI-catalyzed reaction
inhibited DNA competitively and AdoMet in a noncompetitive/mixed manner
with inhibition constants of 3.13 ± 0.05 and 2.78 ± 0.58 nM, respectively. The AdoHcy acted as a competitive
inhibitor of AdoMet in the methylation reaction with
Ki value of 1.55 ± 0.2 nM. The
competitive inhibition of AdoMet by AdoHcy is in accordance with the
previous observations in the cases of M.HhaI and
M.BsuRI with inhibition constants of 2.1 ± 0.1 nM (9) and 0.13 µM (29), respectively. The
AdoHcy inhibition of DNA in the M.MspI-catalyzed reaction
was uncompetitive with inhibition constant of 1.51 ± 0.09 nM. The product inhibition pattern, summarized in Table
III, is consistent with a steady state ordered mechanism in which DNA
has to bind first. This argument requires AdoHcy to form a tight
complex with the enzyme-DNA complex that has been previously reported
(26). The mixed plot is envisaged for a random rapid equilibrium
approach or for a random steady state mechanism; the uncompetitive plot
is not consistent with either of these mechanisms. All the kinetic data
for the M.MspI-catalyzed methylation are consistent and best
fit by the SEQUEN program of Cleland (21). Applying the statistical
criteria of Cleland, the standard deviations are least for the SEQUEN
program. In the presteady state analysis, preincubation of
M.MspI with DNA results in a burst of product upon addition
of AdoMet. The burst is followed by a constant rate of product
formation, which is similar to the catalytic constant measured under
steady state conditions. Thus, starting from an MTase-substrate
complex, the rate constant for product formation is significantly
larger during the first few seconds than the catalytic rate constant.
The simplest interpretation of the data is that a slower step or steps
follow DNA methylation, which is detected as the catalytic rate constant.
By using glutathione S-transferase-M.MspI fusion
protein (GST-M.MspI) and a novel mechanism-based inhibitor,
2-pyrimidinone-1-
-D-2'-deoxifuranoside, Taylor
et al. (27) concluded that the binding of DNA by the MspI methylase occurs in advance of the AdoMet binding.
Furthermore, they conclude that the 4-amino position of the internal
deoxycytidine is critical for the sequence-specific binding to the
enzyme. These findings support our proposal of the kinetic mechanism
for MspI methylase, put forth in the present investigation.
The MspI methylase appears to bind the substrate DNA weakly
in the absence of AdoMet, but after addition of AdoMet, a slow
isomerization of the ternary complex precedes release of the products.
The accumulation of the AdoHcy, which has a higher affinity than the
AdoMet for the M.MspI-DNA complex, leads to formation of a
kinetically significant abortive complex.
Tritium Exchange Catalysis by the M.MspI--
The tritium exchange
reaction catalyzed by the M.MspI indicated that the enzyme
was capable of performing catalysis at the C-5 of the target cytosine
even in the absence of the co-substrate AdoMet. Interestingly, the
M.HpaII, an isoschizomer of the M.MspI, does not
perform this reaction (9). Addition of a nucleophile to C-6 of a
pyrimidine generates a carbanion equivalent at C-5 that upon
protonation by water gives the 5,6-dihydropyrimidine adduct (the
Michael Adduct). Reversal of this reaction can lead to exchange of the
5-H of the pyrimidine for a water proton. The general mechanism of
Michael adduct formation has been demonstrated for five enzymes (TS,
dUMP hydroxymethylase, dCMP hydroxymethylase, m5C-DNA
methyltransferase:M.HhaI, and tRNA-uracil methyltransferase) which catalyze electrophilic displacement reactions at C-5 of the
pyrimidine. Hydrogen exchange has been observed for a number of enzymes
that serve as models for enzymatic reactions involved in the formation
of such intermediates (9, 30, 31). The stereochemical consequences of
5-3H exchange reaction are important, since most enzymes
are stereospecific. If C-5 of one face of the pyrimidine were
protonated to form the adduct, the same proton would be removed upon
reversal. No exchange occurs with several enzymes which may be
explained by one of the two mechanisms. (i) Both faces of the
pyrimidine may be accessible to solvent, albeit to different degrees,
and the protonation and deprotonation may be nonstereospecific. Here,
the observed rate of exchange will be slower than the true rate of
formation/reversal of the dihydropyrimidine adduct. (ii) The reaction
may be a direct counterpart of the enzymatic reaction, in which the
stereochemical addition of the entering solvent proton mimics that of
the normal electrophile of the reaction, and the proton removed
emanates from the original 5-H face of the pyrimidine, and the outgoing proton is always removed from the other. Here, the exchange could equal
that of dihydropyrimidine formation/reversal, and the stereochemistry of the normal reaction could be maintained (30). The rate of tritium
exchange for tRNA-uracil-5-methyltransferase is 100-fold slower than
the methylation reaction (31). With M.HhaI, the kcat of tritium exchange is 7-fold greater than
that for methylation (9). In the case of M.MspI the
kcat of tritium exchange is about 3.3 times that
of methylation. In view of the faster tritium exchange compared with
methylation, a detailed stereochemical understanding of exchange is
required for these m5C-MTases.
The investigation of the kinetic mechanism of M.MspI assumes
significance since this enzyme displayed important differences when
compared with other members of the m5C-MTase family and also in view of
the fact that its isoschizomer M.HpaII does not catalyze the
tritium exchange.