From the Institute of Molecular Biology, State
Research Center of Virology and Biotechnology "Vector," Koltsovo,
Novosibirsk Region, 630559 Russia, the § Department of
Chemistry and Biochemistry, University of California, Santa Barbara,
California 93103, and the ¶ Department of Biology, University of
Rochester, Rochester, New York 14627-0211
Received for publication, December 26, 2002, and in revised form, February 20, 2003
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ABSTRACT |
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We studied the kinetics of methyl group transfer
by the BamHI
DNA-(cytosine-N4-)-methyltransferase (MTase)
from Bacillus amyloliquefaciens to a 20-mer
oligodeoxynucleotide duplex containing the palindromic recognition site
GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the Km for either DNA (100-fold higher)
or S-adenosyl-L-methionine (AdoMet) (20-fold
higher); this indicates that dead-end complexes did not form in the
course of the methylation reaction. The DNA methylation rate was
analyzed as a function of both substrate and product concentrations. It
was found to exhibit product inhibition patterns consistent with a
steady state random bi-bi mechanism in which the dominant order of
substrate binding and product release (methylated DNA,
DNAMe, and
S-adenosyl-L-homocysteine, AdoHcy) was Ado-
Met DNA methylation plays an important role in expression of
genetic information. In various eukaryotes methylation is involved in
regulating transcription, mutation, recombination, parental imprinting,
chromatin structure, and other important cellular events (1). In
prokaryotes, a role in regulating transcription of certain genes has
been established for the Dam
DNA-(adenine-N6-)-methyltransferase
(MTase)1 (reviewed in Ref.
2). For example, it was recently shown that an active dam
gene is crucial for the manifestation of pathogenic properties of
Salmonella typhimurium, as well as a number of other bacteria that inhabit the human intestine (3). The biological role of
other prokaryotic MTases, which mainly belong to the type II class of
modification enzymes, appears to be limited to the protection of
cellular DNA against hydrolysis by their cognate restriction
endonucleases (1), although their wider biological participation in
cellular metabolism is under discussion (4). Thus, the elucidation of
the mechanism of action of DNA MTases is of great interest.
The three known types of prokaryotic MTases are classified according to
their substrate methyl group acceptor: cytosine-C5, cytosine-N4, and
adenine-N6 (3). The amino-MTases are likely to
be more closely related to one another in their mechanism of action
compared with the C5-MTases. Not only do they
have a common modification target, an exocyclic NH2-group,
but they also possess common conserved amino acid sequences in their
major structural/functional motifs (5). Moreover, with the C5-MTases,
the catalytic transfer of the methyl group from donor
S-adenosyl-L-methionine (AdoMet) involves
covalent binding of the enzyme to the target cytosine residue (6). In contrast, (adenine-N6-)- and
(cytosine-N4-)-MTases transfer the methyl group
to the exocyclic NH2-group without formation of a transient
covalent bond. On the other hand, adenine and cytosine residues are
substantially different molecular targets. A number of experimental
data indicate that "flipping" of the target nucleoside out of the
DNA helix is a common property of the reactions catalyzed by all DNA
MTases (7). Since the energetic costs of flipping should differ
substantially for deoxyadenosine and deoxycytidine residues, a
different kinetic behavior of the (adenine-N6-)
and (cytosine-N4-)-MTases might be anticipated.
Earlier it was shown that the rate of the chemical step (transfer of
the methyl group to the target base) differed considerably between the
EcoRI (adenine-N6-) and
HhaI (cytosine-C5)-MTases (8, 9). The EcoRI MTase catalyzes a very rapid methyl group transfer with the
kmeth 300-fold higher than the
kcat. In contrast, the HhaI MTase has
a kmeth that is only slightly higher than
kcat (the rate of product release from the
enzyme). It is not clear at this time what determines this difference;
e.g. is methyl group transfer to an exocyclic NH2-group versus a ring C the determining
factor, or is it the nature of the target base? To approach this
question, we began a study of methylation by the BamHI
(cytosine-N4-)-MTase, which catalyzes methyl
group transfer to the internal cytosine residue in the palindromic
recognition site GGATCC (10). BamHI MTase can also methylate
the modified sequence GGm6ATCC, but not
GGATCm5C) (11). Previously, some aspects of BamHI
MTase function were studied using as substrate SV40 and
bacteriophage To try to reconcile the disparate results from various groups, we
decided to compare the BamHI MTase with the T4 Dam
(adenine-N6-)-MTase, which has been the object
of detailed studies (15-18). Because T4 Dam modifies the adenine
residue in the palindromic sequence GATC, which is contained within the
BamHI target sequence GGATCC, it was possible to use the
same oligonucleotide duplexes to investigate methylation by the two
MTases. Such a comparison is of significant interest because the target
exocyclic amino groups are on different target bases. In this regard,
there appears to be a difference in the kinetic behavior of the
(adenine-N6-) and
(cytosine-N4-)-MTases (17-19). For example, the
rate constant of methyl group transfer from AdoMet to the
(cytosine-N4-) position catalyzed by
BamHI MTase is one order of magnitude lower than that for T4
Dam methylation of the (adenine-N6-) position
(17, 19). In addition, T4 Dam has a higher affinity for substrate DNA
duplex and AdoMet compared with BamHI MTase. However, the
results of pre-steady state experiments showed that both enzymes
catalyze effective transfer of the methyl group to DNA independent of
which substrate was preincubated with the enzyme (18, 19). These
results are consistent with random formation of productive ternary
enzyme-substrate complexes. Thus, despite several important
distinctions in their kinetic parameters, it was possible that
BamHI and T4 Dam MTases share a common kinetic mechanism.
Recently, we carried out a detailed investigation of T4 Dam methylation
of a 20-mer DNA duplex (20). Unexpectedly, contrary to the pre-steady
state experimental results, which indicated random binding of
substrates, the steady state kinetic data best fit a strictly ordered
mechanism. This could be explained by the supposition that, initially,
free T4 Dam is capable of binding randomly to AdoMet and DNA in the
first turnover, but after ternary complex formation the enzyme adopts a
conformation with altered substrate binding capabilities and
specifically adapted to catalysis. During the following turnovers T4
Dam acts according to a strictly ordered reaction mechanism of
substrate binding and product release according to the sequence
AdoMet The present work further compares the BamHI and
T4 Dam MTase reactions. We observed product inhibition patterns with
BamHI that are consistent with a steady state random bi-bi
mechanism in which the dominant order of substrate binding and product
release (methylated DNA, DNAMe, and
S-adenosyl-L-homocysteine, AdoHcy) was
AdoMet Enzymes and
Chemicals--
3H-CH3-labeled
S-adenosyl-L-methionine (15 Ci/mmol; 1 mCi/ml)
was purchased from Amersham Biosciences. AdoHcy and sinefungin were from Sigma. Unlabeled AdoMet (Sigma) was purified further by
chromatography on a C18 reversed-phase column as described previously (21). The synthetic 20-mer duplex used as substrate had the
following structure (the recognition sequence GGATCC is italicized):
5'-CAGTTTAGGATCCATTTCAC-3';
3'-GTCAAATCCTAGGTAAAGTG-5'.
A modified duplex containing N4-methylcytosine
(on both strands) in place of the target Cs (underlined) was
used in the product inhibition studies. It was obtained by complete
M.BamHI in vitro methylation of the unmodified
20-mer using unlabeled AdoMet as a methyl group donor. Oligonucleotides
were synthesized on an Applied Biosystems 380A/380B DNA synthesizer,
and their concentrations were determined spectrophotometrically.
Duplexes were obtained by heating and annealing stoichiometric amounts
of complementary oligonucleotides from 90 to 20 °C over 7-12 h.
BamHI MTase was purified to homogeneity as described
previously (19). Protein concentrations were determined by the Bradford
method (22). These values were in close agreement with those determined
spectrophotometrically at 280 nm (in 6.0 M guanidinium
hydrochloride, 0.02 M phosphate buffer, pH 6.5) from the
known composition and molar extinction coefficients of individual
aromatic amino acid residues (23).
DNA MTase Assay--
The DNA MTase assay was similar to that
previously described (19). Briefly, BamHI MTase reactions
(final volume = 25 µl) were carried out at 37 °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 (21). A low concentration of BamHI
MTase (10 nM) was used in most of the experiments. The
concentrations of AdoMet, DNA duplex, and inhibitors varied according
to the experiment. Reactions were initiated by the addition of
prewarmed BamHI MTase to preincubated mixtures of
[3H-CH3]AdoMet and substrate DNA (with or
without added inhibitor). The reaction times used in steady state
experiments were selected to ensure linear initial velocity conditions;
i.e. during the time of the reaction, terminal product
formation was less than 10% of the initial substrate and added product
inhibitor concentrations. 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). The filters were washed three
times with 0.02 M NH4HCO3, twice
with water, once with 75% ethanol, and then dried. They were counted
in a toluene liquid scintillator. The molar concentrations of
3H-CH3-labeled groups incorporated into DNA
were quantified as described previously (17). 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-labeled
groups incorporated into DNA coincided with the reaction mixture
concentrations of methylatable Ade-residues. All experiments were done
at least twice.
Data Analysis--
Kinetic data were analyzed using the program
Scientist 2.01 (MicroMath®) for 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 shown in Equation 1
Dependence of Initial Methylation Rate on Substrate
Concentration--
Fig. 1 shows the
initial methylation rate as a function of AdoMet and 20-mer duplex DNA
concentrations. It can be seen that both curves have an ordinary
hyperbolic character; saturation of BamHI MTase by both
substrates resulted in a maximum reaction rate. In contrast to T4 Dam
(20), BamHI MTase methylation was not inhibited by high
concentrations of substrate DNA nor was it activated by high
concentrations of AdoMet without visible signs of saturation (20).
The absence of substrate inhibition at concentrations much higher than
Km values for either DNA (100-fold higher) or AdoMet
(20-fold higher) indicates that dead-end complexes did not form in the
course of the reaction. In the case of T4 Dam, inhibition by high
concentrations of substrate DNA was explained by the formation of
dead-end complexes (Dam-DNA and Dam-AdoHcy-DNA), which slow down
progress of the reaction (20). It should be noted that both T4 Dam (21)
and M.BamHI2 can
form dimers. However, they remain as monomers under steady state
conditions, viz. [enzyme] < [DNA] and AdoMet
present in excess.
Inhibition by Reaction Products--
To determine the order of
BamHI MTase substrate binding and product release, we
studied DNA methylation rate as a function of the substrate and product
concentrations (Fig. 2, A-D).
The character of all the double-reciprocal plots were analogous to those obtained earlier with the T4 Dam MTase (20). In summary, AdoHcy
was a competitive inhibitor with regard to AdoMet (Fig. 2A)
and a non-competitive inhibitor with respect to substrate unmethylated
20-mer duplex (D) (Fig. 2B). The other reaction product, fully methylated 20-mer duplex (P), was a non-competitive inhibitor with regard to both AdoMet and unmethylated 20-mer duplex (Fig. 2,
C and D).
To confirm the non-competitive nature of the inhibition by fully
methylated duplex, we also measured the dependence of the reaction
velocity on total DNA concentration. In this experiment, increasing
equimolar amounts of (P) and (D) were introduced in the reaction
mixtures. Analysis of standard equations for competitive and
non-competitive inhibition types (25) showed that the non-competitive inhibition equation predicts that the initial rate will reach a maximum
and then decline with increasing total DNA concentration. As predicted,
the results in Fig. 3 showed a maximum at
[DNA] Models of the Bam Reaction Mechanism--
Scheme 1 (Fig.
4) represents the minimal kinetic scheme
needed to describe all the effects of substrates and products on the reaction rate (Figs. 1-3). The reaction route in this scheme
corresponds to the order of substrate binding and product release
predicted above. Although possible dead-end complexes are included in
this scheme, they do not contain substrate DNA (D) because no reaction inhibition was observed at elevated DNA concentrations (up to 20 µM). It should be noted that unmethylated 20-mer
substrate DNA duplex D is initially converted to the hemi-methylated
product, mD (19), and not to fully methylated duplex, P, which is an inhibitor of the reaction. Therefore, the step EHmD
A complete set of kinetic data, presented in Figs. 1-3 and derived
from additional experiments, was analyzed to determine how well they
fit the kinetic model of Scheme 1. The equations were derived using an
approach developed earlier (26) and are presented in the Supplement at
http://www.jbc.org. It is seen from Scheme 1 kinetic
equation that some of the reaction rate constants
(k3, k5,
k7, see Table II,
Scheme 1 column) can not be determined independently. Table II presents
the results of the most successful and stably reproducible fitting of
the experimental data to Scheme 1 kinetic equation (MSC = 4.29).
Only two individual rate constants, which characterize the interaction
of the free enzyme with AdoMet and the decomposition of this binary
complex, fit Scheme 1 kinetic equation. The remaining constants form
combinations, which perform as independent kinetic and equilibrium
parameters as a part of Scheme 1 kinetic equation. Kinetic and
equilibrium parameters for Scheme 1 were calculated with satisfactory
precision (standard deviation
In parallel, we fit the experimental data to a variant of Scheme 1, where EHP complex formation from complex EH was introduced. Accordingly, Scheme 1 kinetic equation was modified (variant Scheme 1 and equation are not shown). The MSC for the modified scheme had the
same value of 4.29, but all the kinetic constants related to EH complex
conversions, including KEHP, had high standard
deviations (from 10- to 100-fold the mean value). It must be noted that
the calculated mean value of KEHP = 0.5 µM is much higher than the values of
KEP = 0.1 µM and
KESP = 0.06 µM, which were
determined at the same time with a high degree of precision (Table II).
Hence, the elimination of the weak affinity complex EHP from Scheme 1 does not detract from the description of the experimental data, but it
permits determination of the remaining reaction kinetic parameters with
satisfactory standard deviations.
Since earlier pre-steady state kinetic data (19) did not exclude a
random route for the productive complex ESD formation, we attempted to
describe the steady state experimental data by Scheme 2 (Fig.
4), where BamHI MTase random binding with both substrates is
permissible. Scheme 2, with addition of an alternative route of enzyme
binding with substrate, in the order DNA
From the fact that the experimental data can be described by the two
schemes with similar degrees of fitness (MSC1 = 4.29 and MSC2 = 4.33), one could suppose that there is a
preferential route of central complex ESD formation, as in Scheme 1, AdoMet
Similarly, we tried to determine the relative contributions
of possible routes of reaction product release. For this, we introduced into Scheme 2, EHmD
The rate constant for the chemical step methyl group transfer from
AdoMet to DNA is one order of magnitude lower for BamHI MTase, kmeth = 0.085 s
From the relationship between the kcat and
kmeth values, it is possible to estimate an
averaged parameter that characterizes an "effective" (27) rate
parameter for BamHI MTase release from the reaction
products,
k5k7/(k5 + k7) = 0.14 s
Kinetic mechanisms of DNA methylation reactions have been studied only
for a limited number of (adenine-N6-) and
(cytosine-C5-)-MTases; however, comparable studies with (cytosine-N4-)-MTases have not been done. Among
(adenine-N6-) DNA MTases, an ordered reaction
mechanism (where AdoMet is the first substrate bound) was derived for
the EcoRI (28), EcoRV (29), and T4 Dam (20)
MTases. In the case of Ccr MTase (30), an ordered mechanism was
postulated with the enzyme binding to DNA first. A random mechanism of
substrate binding has been reported for the EcoP15I MTase (23).
Finally, a single kinetic mechanism was not found for the
EcaI MTase (24).
(Cytosine-C5) DNA MTases form a covalent binary complex intermediate
with DNA, where subsequent interaction with AdoMet results in transfer
of the methyl group and splitting out of a C5-proton (6). Hence,
an ordered kinetic mechanism with DNA first bound to the enzyme seems
to be the logical pathway. Such a mechanism was shown for the
HhaI (6, 9), MspI (31) and Dnmt1
(mouse) (32) MTases. Recent studies on the HhaI (33) and
DnmT1 (human) (34) MTases have led the authors to conclude
that these enzymes catalyze methylation via a random kinetic mechanism.
These experiments differed from one another in the ranges of substrate
concentrations utilized (6, 9, 33).
The apparent capability of a DNA MTase to randomly bind substrates and
release reaction products does not always agree with the fact that
there is really an ordered kinetic mechanism. We encountered such a
problem studying the T4 Dam reaction mechanism (20). The parameters for
random binding of substrates and release of reaction products did not
agree with the steady state kinetic data, which favored a strictly
ordered mechanism. However, satisfactory agreement could be obtained by
hypothesizing that, initially, free enzyme is capable of random binding
to the substrates. However, once the ternary MTase-AdoMet-DNA complex
is formed, the enzyme acquires an altered conformation. After catalysis
of methyl group transfer from AdoMet to DNA, the enzyme remains in a
conformation that preferentially binds AdoMet, and hence, further
catalytic cycles are carried out according to a strictly ordered
reaction mechanism (20). Such a hypothesis would permit a satisfactory resolution of the apparent conflict between the different
HhaI MTase studies (9, 33).
In conclusion, the kinetic schemes of the reactions catalyzed by the T4
Dam (adenine-N6-)-MTase (30) and the
BamHI (cytosine-N4-)-MTase (this
work) differ significantly. A comparison of the two MTases is
summarized in Table III. Thus, the common
chemical step of the reaction, methyl transfer from AdoMet to a free
exocyclic amino group, is not sufficient to dictate a common kinetic
scheme. Nonetheless, both enzymes follow the same overall
reaction route, AdoMetDNA
DNAMe
AdoHcy
. The M.BamHI
kinetic scheme was compared with that for the T4 Dam
(adenine-N6-)-MTase. The two differed with
respect to an effector action of substrates and in the rate-limiting
step of the reaction (product inhibition patterns are the same for the
both MTases). From this we conclude that the common chemical step in
the methylation reaction, methyl transfer from AdoMet to a free
exocyclic amino group, is not sufficient to dictate a common kinetic
scheme even though both MTases follow the same reaction route.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
DNAs (12) or plasmid pBR322 DNA
(13). At salt concentrations above 120 mM the
enzyme's preference for macromolecular DNA increased (13). This led to
the conclusion that changes in the rate of interaction of
BamHI MTase depended on the position of the recognition site and that longer substrate DNA facilitated linear diffusion of the
enzyme. Study of methylation of an extended substrate DNA showed that a
single binding event was accompanied by the methylation of both strands
in the palindromic recognition site (12), which is contrary to the
behavior of the EcoRI
(adenine-N6-)-MTase (14).
DNA
DNAMe
AdoHcy
.
DNA
DNAMe
AdoHcy
. Although the overall
reaction is similar to that for T4 Dam, the two kinetic schemes differ;
viz. with respect to an effector action of substrates and in
the rate-limiting step of the reaction. Thus, a common chemical step in
the methylation reaction, methyl transfer from AdoMet to a free
exocyclic amino group, is not sufficient to dictate a common
kinetic scheme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
where Yobs is the weighted mean of the
observed data, n is the number of points,
wi is the weight applied to each point, and
p is the number of parameters. The model that has the
largest MSC is by definition the best or most appropriate model.
(Eq. 1)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (7K):
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Fig. 1.
Methylation rates for 20-mer duplex by
BamHI MTase versus substrate concentration.
Reaction mixtures contained BamHI MTase, 10 nM,
AdoMet, 20 µM (A); BamHI MTase, 10 nM, DNA, 1 µM (B).
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Fig. 2.
Double-reciprocal plots analyzing product
inhibition of the methylation reaction catalyzed by
BamHI MTase. A and B,
inhibition by AdoHcy; C and D, inhibition by
fully methylated 20-mer DNA duplex. The DNA concentration was fixed at
150 nM (A, C); the AdoMet
concentration was fixed at 2 µM (B,
D). The BamHI MTase concentration was 10 nM in all cases. The concentrations of inhibitor (AdoHcy or
DNAMe) are given in the insets. Least squares
linear regressions for the reciprocals of the reaction velocity (1/V)
versus reciprocal substrate concentration (1/AdoMet or
1/DNA) (at fixed concentrations of AdoHcy or methylated 20-mer DNA
duplex) are represented by the solid lines.
0.6 µM and then declined at higher
concentrations. Thus, as for the T4 Dam MTase, these results
(summarized in Table I) are consistent
with a steady state, BamHI MTase substrate binding, and
product release that obey the scheme
AdoMet
DNA
DNAMe
AdoHcy
.
View larger version (9K):
[in a new window]
Fig. 3.
BamHI MTase methylation rate as a
function of total DNA concentration. BamHI and AdoMet
concentrations were fixed at 10 nM and 10 µM,
respectively; input substrate (unmethylated 20-mer) and inhibitor
product (fully methylated 20-mer) duplexes were kept at a 1:1 ratio
over the entire range of concentrations.
Product inhibition analysis of reaction catalyzed by the M.BamHI
EH + mD is
irreversible. Scheme 1 contains only the dead-end complexes EP and ESP
but not a complex EHP. This will be discussed further below.
View larger version (13K):
[in a new window]
Fig. 4.
Alternative schemes for
BamHI MTase methylation of the 20-mer DNA duplex.
E, BamHI MTase; S, AdoMet; H, AdoHcy; D, (20-mer)
unmethylated DNA duplex; mD, hemi-methylated DNA duplex; P, fully
methylated DNA duplex.
10%) except for the rate constant
k
1, which was determined with a significant
standard deviation. Additional support for this fitting is the fact
that the curve in Fig. 3 is perfectly described by Scheme 1 kinetic
equation at S = 10 µM, H = 0 µM,
and [D]i = [P]i. The maximum for the
equation is D2max = KEP
(k1S + k
1)/Q3. If we put into this expression the values of parameters from Table II and [S] = 10 µM, we obtain D2max = 0.098 ((0.039) (10) + 0.019)/0.41 = 0.098, where Dmax = 0.31 µM. Multiplied by factor of 2, [DNA]max = 0.62 µM, which coincides perfectly with the
experimentally observed maximum value.
Kinetic parameters of M.BamHI calculated for steady state ordered bi-bi
(Scheme 1) and random two-route (Scheme 2) mechanisms
AdoMet
, had an MSC = 4.33; the calculated values of the kinetic and equilibrium constants
are presented in Table II. Scheme 2 kinetic equation contains several
new constants that are not present in the first equation. Table II
shows that values for most of the parameters, which characterize
identical reaction steps or their combinations (and can be determined
for the both schemes), coincided within standard deviation limits.
DNA
. A fraction of this pathway in the whole reaction
velocity is determined by the expression (26) in Equation 2.
Using the program Scientist, we have calculated f1 values for all
experimental points (not shown). With the exception of a small number
of them, where the concentration of DNA was significantly higher than
that of AdoMet, the f1 values were in the range 0.7-0.98. In
particular, where substrate concentrations were close to their Km values (KmDNA = 0.2 µM and KmAdoMet = 1.22 µM), the f1 values were approximately 0.95. Additional support for the reaction route where enzyme binds AdoMet first comes
from the coincidence in the two-step parameter
Q3 for the both schemes. This parameter was
found as a result of the fitting for Scheme 1 and was calculated for
Scheme 2 using the calculated values for constants
k3 and k
(Eq. 2)
3. Taken
together, the results of fitting are best described by a dominant route
of reaction in which the sequence of substrate binding is
AdoMet
DNA
.
EmD
E + mD. However, it was found that this
resulted in such large increases in standard deviations of the
calculated parameters that it was impossible to approximate the
relative effectiveness of the corresponding routes. Evidently, the
reason for such uncertainty is due to an insufficient amount of
experimental data. However, on the basis of the graphic analysis and
good fitting of the data to Scheme 2 kinetic equation (with one route
of product release), the dominant route of product release appears to
be DNAMe
AdoHcy
. This hypothesis is consistent with
the fact that, as with most other enzymes, the order of the product
release is a "mirror reflection" of the order of substrates binding
(25).
1 (averaged
value of two values determined in Ref. 19) versus kmeth = 0.56 s
1 for T4 Dam (18).
Yet, the value of kcat = 0.052 s
1
is ~3-fold higher than for T4 Dam (17). Earlier it was shown that
kmeth is ~1.5-2-fold greater in comparison
with kcat (0.053 s
1) for the
BamHI MTase (19). In addition, BamHI MTase
catalyzed a burst in the reaction. These observations were consistent
with the rate-limiting step being that of product dissociation.
However, a more detailed analysis presented in this work leads us to
alter that conclusion.
1 (for details
see Supplement 3 at http://www.jbc.org). Evidently, neither
constant from this expression can be smaller than the effective parameter value. Hence, for the sequence of steps in Reaction 1,
the rate constant of the chemical step
kmeth = 0.085 s
1 has the lowest
value, so this step limits the overall reaction rate. In contrast, the
release of AdoHcy from the complex EH is rate-limiting for T4 Dam (20)
(Table II).
DNA
DNAMe
AdoHcy
.
Differences between kinetic properties of M.BamHI MTase and T4 Dam
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ACKNOWLEDGEMENTS |
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The authors thank A. V. Zinoviev for technical assistance and F. V. Tuzikov for the helpful discussions.
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FOOTNOTES |
---|
* This work was supported by the Russian Foundation for Basic Research Project 01-04-49869 and United States Public Health Service Grants R03 TW05755 from the Fogarty International Center and GM29227 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains kinetic equations for Schemes 1 and 2 and an estimation of an effective rate parameter for BamHI
MTase release from the reaction products.
To whom correspondence should be addressed. Tel.:
585-275-8046; Fax: 585-275-2070; E-mail:
modDNA@mail.rochester.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M213213200
2 E. G. Malygin, V. V. Zinoviev, A. A. Evdokimov, W. M. Lindstrom, Jr., N. O. Reich, and S. Hattman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MTase, methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; DNAMe, methylated DNA; MSC, model selection criterion.
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REFERENCES |
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