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J. Biol. Chem., Vol. 277, Issue 1, 279-286, January 4, 2002

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Bacteriophage T4 Dam DNA-[N⁶-adenine]Methyltransferase

KINETIC EVIDENCE FOR A CATALYTICALLY ESSENTIAL CONFORMATIONAL CHANGE IN THE TERNARY COMPLEX^*

Alexey A. Evdokimov, Victor V. Zinoviev, Ernst G. Malygin, Samuel L. Schlagman^§, and Stanley Hattman^§¶

From the  Institute of Molecular Biology, State Research Center of Virology and Biotechnology Vector, Novosibirsk 630559, Russia and the ^§ Department of Biology, University of Rochester, Rochester, New York 14627-0211

Received for publication, September 13, 2001, and in revised form, October 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N⁶-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-L-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA^Me and S-adenosyl-L-homocysteine, AdoHcy) release was AdoMetDNADNA^MeAdoHcy. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA^Me dissociates relatively rapidly (k_off = 1.7 s¹) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k_off = 0.018 s¹), indicating that its release is the rate-limiting step, consistent with k_cat = 0.015 s¹. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

DNA methyltransferases (MTases)¹ are involved in a variety of important cellular functions in both prokaryotes and eukaryotes (1-3). In addition, DNA MTases are excellent subjects for detailed studies of specific protein-DNA interaction because they are highly specific and have a comparatively simple organization. Elucidating the kinetic mechanism of the reactions catalyzed by these enzymes still remains an important problem to investigate in the area of biological DNA methylation. Kinetic schemes have been proposed for HhaI (4-7), MspI (8), human Dnmt1 (9), and murine Dnmt1 (10) [C⁵-cytosine]MTases, and for EcoRI (11, 12), EcaI (13), TaqI (14), EcoRV (15), and Type III EcoP15I [N⁶-adenine]MTases (16). All of these enzymes exhibit a sequential bi-bi mechanism; however, they differ with respect to the order of substrate binding and rate-limiting step. For instance, whereas the rate of methyl group transfer is at least 300-fold faster than the rate of dissociation of the products for EcoRI (12), in contrast, the rate of methyl group transfer is the rate-limiting step in the TaqI methylation reaction (14). Furthermore, several different modes of MTase binding to the substrates DNA (17, 18) and AdoMet or its analogs (5, 19) have been distinguished. In addition, changes in enzyme conformation (isomerization) associated with binding substrate DNA and/or AdoMet may also influence the reaction kinetics. Allosteric activation by methylated DNA was observed with human Dnmt1 MTase (9, 20). The binding of two AdoMet molecules was reported for the EcoDam (21) and PvuII MTases (22, 23), and an effector role was suggested for the second AdoMet molecule. The precise binding stoichiometry of AdoMet, as well as its double role as methyl donor and allosteric effector, remains to be fully characterized for T4 Dam (24, 25). Thus, in contrast to the apparently universal ternary structure of the catalytic domains (23, 26-29), the existing biochemical data are complex and not clear enough for generalizations to be made concerning the kinetics of the reactions catalyzed by the different DNA MTases. Therefore, further investigation is required.

The Dam DNA-[N⁶-adenine]MTase encoded by phage T4 catalyzes methyl group transfer from AdoMet to the N⁶ position of an Ade residue in the palindromic sequence, 5'-GATC-3' (30). T4 Dam belongs to the large family of -group type II DNA MTases (31), in which there are at least 50 members known today and almost half of them are [N⁶-adenine] GATC-specific isoschizomers. Furthermore, the enzymes that modify the N⁶-amino nitrogen of adenine share not only the nine conserved motifs, but also possess striking similarity in their target recognition domains, although they have different targets; e.g. GATC, GANTC, GATATC, and non-palindromic CATCC (28). Such homology may, in turn, indicate that a considerable amount of similarity exists in their three-dimensional structures, as well as in their mechanism of action. Recently, more progress has been made in the study of this family of enzymes; the first crystal structure for the GATC-specific DpnM MTase complexed with AdoMet was reported (28), and an extensive biochemical investigation of the GATATC-specific EcoRV MTase was performed (15, 32-35).

Previously, catalytic turnover and Michaelian constants were obtained for the T4 Dam methylation reaction using either polymeric 5-hydroxymethylcytosine-containing T4 gt gt (unglucosylated) dam (unmethylated) DNA (hmCyt-DNA) or a set of synthetic oligonucleotide duplexes containing one or more defect(s) or substitution(s) in the target sequence (36-38). In this communication, we report a detailed steady state kinetic analysis of the methylation of a synthetic 20-mer oligonucleotide duplex catalyzed by T4 Dam; and, we propose a kinetic reaction scheme for this member of the -group GATC-family of DNA MTases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

Enzymes and Chemicals-- [³H-CH₃]S-adenosyl-L-methionine (15 Ci/mmol; 1 mCi/ml) was purchased from Amersham Biosciences. S-adenosyl-L-homocysteine (AdoHcy) and sinefungin were from Sigma. Unlabeled AdoMet (Sigma) was purified further by chromatography on a C₁₈ reversed-phase column as described previously (36). The synthetic 20-mer duplex used as substrate had the following sequence shown in Structure I. 


A modified duplex containing N⁶-methyladenine in the recognition sequence GATC (underlined) in both strands was used in the product inhibition studies. Oligonucleotides were synthesized on an Applied Biosystems 380A/380B DNA synthesizer, and concentrations were determined spectrophotometrically. The duplexes were obtained by heating and annealing complementary oligonucleotide chains from 90 to 20 °C over 7-12 h. T4 Dam was purified to homogeneity as described previously (36). The protein concentration was determined by the Bradford method (39), which yielded values in close agreement with those determined spectrophotometrically at 280 nm from the known composition and molar extinction coefficients of individual aromatic amino acid residues in pH 6.5, 6.0 M guanidinium hydrochloride, 0.02 M phosphate buffer (40).

DNA MTase Assay-- DNA MTase assays were similar to that previously reported (38). T4 Dam reactions were carried out at 25 °C in buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 0.2 mg/ml bovine serum albumin (36). A low concentration of T4 Dam (1 nM) was used for most experiments to increase the accuracy of the calculation of the initial reaction velocities by reducing the influence of any burst in product formation (37). The concentrations of AdoMet, substrate DNA, and inhibitors varied according to the individual experiment. The reactions were initiated by addition of prewarmed T4 Dam to preincubated mixtures of [³H-CH₃]AdoMet and substrate DNA (with or without inhibitors; final volume is 25 µl). The reaction time used was selected to ensure initial velocity conditions; i.e. product formation was less than 15% of the initial substrate and added product inhibitor concentrations during the time of the reaction. At appropriate intervals, an aliquot (15 µl) was withdrawn from each mixture and spotted on a DE81 anion-exchange filter disc (Whatman, 1.5 cm). Filters were washed three times with 0.02 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] groups incorporated into DNA were quantified as described previously (41). The validity of the quantification procedure was confirmed under complete methylation conditions (about 1 h at a 1:2 enzyme/substrate ratio), where the calculated concentrations of [³H-CH₃] groups incorporated into DNA coincided with the reaction mixture concentrations of methylatable Ade residues. All experiments were done at least twice.

Data Analysis-- Kinetic data were analyzed using the program Scientist 2.01 (MicroMath^®) for non-linear regression analysis. We used the statistical model selection criterion (MSC) recommended by program developers to determine the goodness of fit for each kinetic model. The MSC is defined by the formula in Equation 1,

(Eq. 1)

where n is the number of points, w_i are the weights applied to each point, Y_obs is the weighted mean of the observed data and p is the number of parameters. The model that has the largest MSC value is by definition the best or most appropriate model.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

The Reverse of the Methylation Reaction Is Not Detectable-- The ability of T4 Dam to catalyze the transfer of a methyl group from methylated DNA to AdoHcy was tested using an [N⁶-³H-methyl]adenine-containing 20-mer DNA duplex at a concentration of 200 nM. Reactions were performed in the presence of 50 nM T4 Dam and 0, 5, or 50 µM AdoHcy. No tritium loss from the [N⁶-³H-methyl]adenine containing 20-mer was observed over a period of 270 min (data not shown). This indicated that the reverse reaction is at least 500-fold slower than the forward one. Therefore, the transfer of the methyl group from AdoMet to DNA can be considered irreversible for the T4 Dam MTase; this has been previously shown for the HhaI (4) and MspI (8) MTases.

Initial Velocity Dependence at Moderate Substrate Concentration-- Initial velocities (V) were determined at various concentrations of the substrates, [³H-CH₃]AdoMet and 20-mer duplex DNA. The substrate concentrations used in these experiments were up to 5-fold above K_m for AdoMet (K_m AdoMet = 490 nM) and 15-fold above K_m for DNA (K_m DNA = 6.3 nM) (37). As shown in Fig. 1, both double-reciprocal plots gave a series of straight lines that intersected to the left of the 1/V axis, which rules out a ping-pong bi-bi mechanism. An ordered rapid-equilibrium mechanism is also unlikely because the double-reciprocal plot lines should have intersected at the 1/V axis for the second substrate that binds. In addition, secondary plots of the slopes and 1/V intercepts versus reciprocal concentrations of substrates were approximately linear (not shown), permitting calculation of conventional kinetic parameters. In accordance with graphical predictions, the experimental data fit an equation that corresponds to either a steady state-ordered bi-bi or a rapid-equilibrium random bi-bi mechanism (42) according to Equation 2.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Double-reciprocal plots of the initial reaction velocity versus substrate concentration for methylation by T4 Dam. A, varying the AdoMet concentration with the 20-mer DNA duplex concentrations at the levels shown in the inset. B, varying the 20-mer DNA duplex concentration with the AdoMet concentrations at the levels shown in the inset. The T4 Dam MTase concentration was 1 nM in all cases. 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 the other substrate) are represented by solid lines.

(Eq. 2)

Because the conversion step (k_meth = 0.56 s¹) in the T4 Dam methylation reaction (43) was much faster than the catalytic turnover constant (k_cat = 0.015 s¹), a rapid equilibrium random mechanism appears to be ruled out. Results of product inhibition studies below support this conclusion.

Product Inhibition Analysis-- Product inhibition studies are commonly performed to determine whether there is a preferential order of substrate binding for a particular multiple substrate reaction (44). We used this approach to determine whether T4 Dam first binds substrate DNA or AdoMet (Fig. 2 and Table I). We found that AdoHcy was a competitive inhibitor with respect to AdoMet (Fig. 2A) and a non-competitive inhibitor with respect to 20-mer DNA duplex (Fig. 2B); this is consistent with previous results (36). Similar inhibition patterns were also obtained with sinefungin, another non-reactive AdoMet analog (data not shown). The other reaction product, fully methylated 20-mer DNA duplex, exhibited non-competitive inhibition with respect to both AdoMet and unmethylated 20-mer DNA duplex (Fig. 2, C and D). Secondary plots of the slopes and 1/V intercepts versus concentration of inhibitor were approximately linear (not shown). Thus, these product inhibition patterns (Table I) are consistent with a steady state-ordered bi-bi mechanism (44), in which the substrate binding and product release order are AdoMetDNADNA^MeAdoHcy.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Double-reciprocal plots analyzing product inhibition of the methylation reaction catalyzed by T4 Dam. 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 T4 Dam MTase concentration was 1 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 solid lines.

Studies at High Substrate Concentrations-- Generally, DNA MTases may form binary complexes with either of their reaction substrates. Binding of AdoMet in the absence of substrate DNA has been confirmed, in particular by co-crystal structures (5, 19, 23, 28) and by copurification of MTase with tightly bound AdoMet (45-47). On the other hand, MTases are capable of recognizing and binding at specific DNA sequence(s) and flipping the target base in the absence of AdoMet or its non-reactive analogs (17, 34, 48, 49); although, as a rule, AdoMet (or its non-reactive analogs) increases the enzyme's affinity for substrate DNA (13, 17, 21, 50-52). However, DNA MTases can also form stable, non-functional (dead-end) enzyme-product-substrate ternary complexes, as has been observed for the MTase-AdoHcy-DNA complex of HhaI (53). Thus, if the initial concentration of the second substrate to bind were sufficiently high, a steady state-ordered reaction (which has a specific order of substrate binding and product release) would show inhibition of the initial reaction velocity due to the formation of non-productive binary and/or dead-end ternary complexes (54). In contrast, a rapid equilibrium random bi-bi mechanism should show no substrate inhibition when the initial concentration of either (or both) substrate(s) is high (54). The results in Fig. 3 show a strong inhibition in T4 Dam initial reaction velocity at high concentrations of substrate 20-mer DNA duplex. This indicates that T4 Dam does not obey a rapid equilibrium random bi-bi mechanism. Rather, the data are consistent with a steady state-ordered bi-bi mechanism, as we had concluded from the product inhibition analysis.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Substrate inhibition of the T4 Dam methylation reaction by high concentrations of 20-mer oligonucleotide DNA duplex. The concentrations of T4 Dam and AdoMet were fixed at 1 nM and 2 µM, respectively. Increasing the concentration of the 20-mer DNA duplex above 0.2 µM led to a progressive decrease in the initial methylation velocity. The solid line was calculated using the equation for Scheme 2 (Fig. 5); the kinetic parameters are presented in Table III. The inset shows the initial portion of the curve fitted to a conventional Michaelian dependence.

In contrast to the results with the substrate 20-mer DNA duplex, increasing the concentration of AdoMet led to a progressive stimulation in the reaction rate (Fig. 4). Whereas the initial portion of the concentration dependence curve corresponded approximately to a conventional hyperbolic dependence (Fig. 4, inset), saturation was never achieved. In fact, the rate of the reaction was linearly proportional to the AdoMet concentration up to 30 µM (60-fold above the K_m for AdoMet). We will present possible explanations of this unexpected effect in the "Discussion."

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Substrate activation of the T4 Dam methylation reaction by high concentrations of AdoMet. The T4 Dam concentration was at 1 nM and the DNA duplex concentration was at 200 nM. The solid line was calculated using the equation for Scheme 2 (Fig. 5); the kinetic parameters are presented in Table III. The inset shows the initial portion of the curve fitted to a conventional Michaelian dependence.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

The HhaI (4) and MspI (8) DNA MTases have been analyzed for their ability to perform the reverse of the methylation reaction; e.g. transferring the methyl group from [methyl-³H]DNA to AdoHcy to form [methyl-³H]AdoMet. Both enzymes were incapable of performing the reverse catalytic reaction, and we have shown here that T4 Dam is also unable to do so. Therefore, the T4 Dam-catalyzed methylation reaction appears to be irreversible, which has facilitated further kinetic analysis.

Preferential Pathway of the T4 Dam-catalyzed Reaction-- The product inhibition patterns obtained for the T4 Dam MTase (Fig. 2, Table I) are consistent with a steady state-ordered bi-bi mechanism, in which the order of substrate binding and product release are AdoMetDNADNA^MeAdoHcy. However, the stimulatory effect of high AdoMet concentrations on DNA methylation rate indicates that the mechanism is more complex; that is, the release of AdoHcy from the MTase-AdoHcy complex appears to be associated with the binding of AdoMet in a concerted event (discussed below). In a previous study, polymeric T4 gt gt (unglucosylated) dam⁺ hmCyt-DNA (previously methylated in vitro by T4 Dam) was observed to be a competitive inhibitor of unmethylated substrate hmCyt-DNA (36). In the experiments reported here; however, methylated 20-mer DNA duplex was a non-competitive inhibitor of the unmethylated 20-mer DNA duplex DNA. The difference in the nature of the inhibition can be explained, as a formal analysis indicates (not shown here), by a preferentially processive methylation mechanism of polymeric DNA, in contrast to a distributive methylation mechanism of the 20-mer duplex. This question is currently under investigation.

The preferential order of substrate binding proposed for T4 Dam is identical to that found for two other [N⁶-adenine]MTases, EcoRI (11), and EcoRV (15). The TaqI (14) and EcoP15I (16) MTases exhibit a random mechanism for substrate binding and product release. An ordered bi-bi mechanism with DNAAdoMet order of substrate binding is usually assigned for the [C5-cytosine]DNA MTases; e.g. for the HhaI (4, 6), MspI (8), and murine Dnmt1 MTases (10). However, a random mechanism has been proposed for the human Dnmt1 MTase (9) and HhaI (7).

Rapid kinetics measurements (in decisecond and second time intervals) of methylation of the 20-mer duplexes revealed that T4 Dam (E) preincubated with AdoMet (S) prior to the addition of DNA (D) had a 4-fold faster chemical conversion rate (k_meth = 0.56 s¹) than T4 Dam preincubated with DNA prior to the addition of AdoMet (k_meth = 0.14 s¹) (43). We proposed that the ternary complex formed during interaction of ED with S to form EDS is not fully enzymatically competent and that it must isomerize to form a productive complex ESD before the chemical conversion reaction can take place. However, because the chemical step is not the rate-limiting step in the reaction (43), the 4-fold lower rate of chemical conversion cannot account for the nearly 10-fold lower k_cat (Fig. 3). Therefore, some step subsequent to chemical conversion, and one that is involved in product release, must be responsible for the reduction in k_cat observed at elevated concentrations of DNA. Most likely, this step involves the production of dead-end EHD ternary complexes (H is AdoHcy). In this regard, substrate inhibition of the methylation reaction by AdoMet was observed for M.EcoP15I (16); it was suggested that this could be due to the formation of a dead-end ternary enzyme-(DNA^M)-AdoMet complex, provided that release of product DNA^M is the rate-limiting step.

A Special Role of AdoMet in the T4 Dam Methylation Reaction-- Increasing the concentration of substrate AdoMet did not lead to the reaction rate approaching a plateau, as would be expected for a hyperbolic dependence. Instead the reaction rate increased almost linearly with the concentration of AdoMet (Fig. 4). These results can be explained if T4 Dam were to possess two AdoMet binding sites. The first is the catalytic site with a high affinity for AdoMet, and the second is an allosteric site with a low affinity for AdoMet. When AdoMet binds to the second site, the enzyme undergoes an allosteric rearrangement, resulting in an increased reaction rate, due to its higher catalytic efficiency. This has been suggested for both the EcoDam (21) and PvuII (22) MTases. In this regard, however, using equilibrium dialysis to study the stoichiometry of binding, it was calculated that T4 Dam binds 1.35 ± 0.27 molecules of AdoMet (24). Although this was taken to indicate the binding of a single AdoMet, the precise stoichiometry is really not clear and a reanalysis is warranted.

The maximum concentration of AdoMet used in the experiment represented in Fig. 4 was 30 µM. In a separate experiment, we increased the AdoMet concentration up to 150 µM (data not shown), and still observed an approximately linear increase in the reaction rate. These results suggested that an additional explanation for the effect of AdoMet was necessary. Catalase is another enzyme with reaction rate characteristics similar to those of T4 Dam; e.g. it does not exhibit any tendency to saturate at high concentrations of substrate (55). If product release is the rate-limiting step of the reaction, then this type of behavior may be explained by the binding of substrate and release of product occurring in a concerted event. Our previous results showed that product release is, in fact, the rate-limiting step in the T4 Dam methylation reaction (43). We suggest that in the case of T4 Dam, such a concerted event can be represented as follows in Reaction I,

where AdoHcy is released last and AdoMet binding occurs first. It corresponds formally to a mechanism proposed by Ogura (56). Because enzymatic reactions proceed via enzyme-substrate complexes, we also must propose the existence of a short-lived intermediate complex, EHS. However, this type of conversion can occur only if T4 Dam has two AdoMet binding sites possessing negative cooperativity.

Models of the T4 Dam Reaction Mechanism-- Scheme 1 in Fig. 5 represents the minimal kinetic scheme needed to describe all the affects of substrates and products on the reaction rate. The main route in this scheme has a substrate binding and product release order of AdoMetDNADNA^MeAdoHcy. This corresponds to the order predicted from the product inhibition and effect of high substrate concentration studies. All of the possible dead-end complexes are included in this scheme. It should be noted that substrate unmethylated 20-mer DNA duplex, D, is initially converted to the hemi-methylated product, mD, and not to the completely methylated duplex, P, which is an inhibitor of the reaction. Therefore, the step, EHmD  EH + mD, is irreversible. Furthermore, the dead-end complex EHP is introduced into the scheme, because mD and P are not identical and behave differently in the reaction. Finally, to account for the linear increase in the reaction rate that coincides with the increase in AdoMet concentration, we included the following reversible step in Reaction II.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Alternative schemes for T4 Dam methylation of the 20-mer DNA duplex. E, free T4 Dam; F, an isomerized form of T4 Dam; S, AdoMet; H, AdoHcy; D, (20-mer) unmethylated DNA duplex; mD, hemi-methylated DNA duplex; P, fully methylated DNA duplex.

A complete set of kinetic data, derived from the experimental curves in Figs. 1-4, was analyzed to determine how well they fit the kinetic model of Scheme 1 (Fig. 5). Kinetic analyses of the reactions catalyzed by DNA MTases are generally carried out either qualitatively or use a set of simplified kinetic equations (4, 8, 9, 11, 13). Such simplifications can result in the loss of kinetic information or its misrepresentation. Because of the peculiarities of our kinetic data, we first tried to analyze them simultaneously using an equation (see "Appendix"), representing what occurs in Scheme 1 (Fig. 5). This equation was derived using an approach developed earlier (57). We began the calculations using information previously obtained. For example, the value of k₄ was set equal to k_cat (0.015 s¹). The dissociation constants of the T4 Dam-AdoMet and the T4 Dam-AdoHcy complexes had been previously determined from fluorescence quenching studies to be 0.27 and 2.3 µM, respectively (58). The K_d values determined by gel shift experiments for complexes between T4 Dam and specific 20-mer DNA duplex were 43 nM in the absence of AdoMet and 17 nM in the presence of AdoMet (52). These values were used to calculate the rate constants for the dissociation of the complexes.

In the framework of steady state kinetics, the sequence of the two monomolecular conversions shown in Reaction III,

is determined by one kinetic parameter, Q₀ = k₀ k₃/(k₀ + k₃). Depending on the value of k₃, the value of Q₀ must be k₀. Therefore, for a first approximation of the value of Q₀, we used 0.56 s¹, the k₀ value (which is the chemical step of the reaction) obtained by rapid quench analysis (43). In addition, according to Scheme 1, the k₂ and k₂ values can not be determined separately; however, in the kinetic equation, the value of Q₁ = k₀k₂/(k₀ + k₂). Furthermore for the cycles of complex formation, we used the thermodynamic limitation in which k₅ = k₁k₅k₄/(k₁k₄).

The MSC value of 3.75 (Table II) for Scheme 1, using the specific constraints k₁ = 0.27 k₁, k₂ = 0.017 k₂ and k₄ = k₄/2.3, was an adequate fit. However, the MSC for Scheme 1 significantly improved when these constraints were eliminated (MSC = 4.37). Thus, according to the MSC values, the steady state-ordered bi-bi model satisfactorily fits all of the experimental data. This agrees with our product inhibition analysis (Fig. 2, Table I), and it suggests a steady state-ordered bi-bi mechanism.

However, there are several inconsistencies between the values of some of the kinetic constants determined using Scheme 1 and those experimentally determined previously. The largest discrepancy is between the dissociation constants of enzyme-ligand complexes. For instance, gel shift results indicated a K_ED = 0.047 µM (52). However, when the kinetic data was incorporated into Scheme 1, a very high value for K_ED (>1000 µM) was obtained; this value was independent of the initial parameters. Furthermore, the high K_ED value suggests that free T4 Dam, which enters the steady state reaction cycle, does not form an enzyme-DNA complex. In contrast, pre-steady state kinetic results showed that T4 Dam preincubated with DNA carried out a productive methylation reaction when AdoMet was added. However, the rate of the chemical step for this reaction was 4-fold slower than the rate that occurs when enzyme was preincubated with AdoMet (before the addition of DNA) (43). Thus, a MTase-DNA complex can be formed by free T4 Dam entering the steady state reaction cycle. This contradicts the results obtained using Scheme 1. In addition, the ratio k₄/k₄ from Scheme 1 (the dissociation constant of the MTase-AdoHcy complex) was surprisingly low (~0.3 µM), which does not agree with the value of 2.3 µM measured by fluorescence titration of T4 Dam by AdoHcy (58). Finally, the ratio k₁/k₁ (the dissociation constant of the MTase-AdoMet complex in Scheme 1) is ~1 µM. This indicates that the relative affinity of T4 Dam for AdoMet and AdoHcy during catalysis (determined using Scheme 1) is the reverse of that found under non-catalytic conditions (determined by ligand binding measurements).

AdoMet binding to T4 Dam is known to induce an allosteric alteration in the T4 Dam conformation, as demonstrated by tryptophan fluorescence quenching analysis (58). In addition, fluorescence studies of 2-aminopurine-substituted duplexes (containing the GATC target site) indicated that the specificity of T4 Dam for the DNA strand containing the adenine base in the GATC sequence, increases significantly after it binds AdoMet (25). This suggested that T4 Dam undergoes isomerization after AdoMet binding, which alters the way that it interacts with DNA. Thus, to explain the inconsistencies in Scheme 1, we suggest that after AdoMet binds, T4 Dam undergoes isomerization in the ternary complex to a form that has altered catalytic and binding characteristics. Furthermore, we suggest that it is this new form of the enzyme, F, that acts in the catalytic cycle. Therefore, we propose Scheme 2 (Fig. 5) in which the initial enzymatic forms E, ED, and ES enter the catalytic cycle via an isomerization step, resulting in form FSD. The rest of the catalytic reaction cycle proceeds using the F-form of the enzyme. The kinetic equation corresponding to Scheme 2 includes additional terms (not found in Scheme 1) that take into account the interaction of the initial form of T4 Dam, E, with the substrates, and how these ED and ES forms enter the steady state cycle. The dissociation constants K_ES, K_ED, K_FSD of complexes ES, ED, and FSD were assigned the values that had been previously determined (Table III and Refs. 52, 58).

Our calculations show that the addition of these added steps in the interaction between T4 Dam and its substrates (Fig. 5, Scheme 2) produce only small effects on the estimated values of the overall kinetic parameters of the reaction. The MSC value for Scheme 2 is virtually identical to that of Scheme 1 (Table II). Thus, the hypothesis that T4 Dam isomerizes after interacting with its substrates and that it is this isomerized form of the enzyme that enters the catalytic cycle is feasible. We suggest that Scheme 2, or a slight modification of it, is the kinetic pathway used by T4 Dam in methylating the 20-mer duplex.

According to Scheme 2, the following sequence of events occurs. Free T4 Dam, initially in form E, interacts randomly with AdoMet and DNA to form binary complexes, ES and ED. These complexes interact with the second substrate to form the ternary complex, FSD, in which T4 Dam has isomerized to state F. Form F is significantly different from the initial conformational state E and is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, the methylated duplex dissociates relatively rapidly (k₃ = 1.7 s¹). The dissociation of the second product, AdoHcy, appears to be the rate-limiting step of the reaction and proceeds with k₄ = 0.018 s¹; this agrees with the value of k_cat = 0.015 s¹ determined previously (37). The release of AdoHcy from the FH complex can occur by two pathways. The first involves release of AdoHcy, while the enzyme remains in the isomerized F form, which preferentially binds AdoMet to form an FS complex. DNA then binds this FS complex, and the catalytic cycle continues. This proposed mechanism is in contrast to the classical Iso-mechanisms (55), in which the enzyme converts back to its initial conformational state following release of substrate and prior to the start of a new catalytic cycle.

The second pathway is one in which AdoHcy release and AdoMet binding occur as one concerted event, and the resulting enzyme-AdoMet complex remains in the F conformation ready to bind DNA and continue catalysis. Although the exact mechanism of this concerted event has not been determined, it has important biological consequences. The AdoMet concentration in Escherichia coli, reported to be in the range of 30-300 µM (59) or 300-500 µM (60), is 1-2 orders of magnitude greater than the K_d of the T4 Dam-AdoMet complex (1 µM). Therefore, the velocity of the T4 Dam methylation reaction in vivo would be predicted to be much faster than the reaction rates observed in vitro at moderate substrate concentrations. However, an upper limit does exist for the reaction rate. The Q₀ value (0.42 s¹, see Table III) is 20-fold faster than the k₄ value (0.018 s¹), the rate-limiting step of the reaction. Therefore, the increase in the reaction velocity will be proportional to increasing AdoMet concentration until its rate reaches the Q₀ value. After this point, the rate of the chemical reaction, rather that the rate of release of AdoHcy, becomes the rate-limiting step of the reaction. Nevertheless, this indicates that in vivo T4 Dam-dependent steady state catalysis may be 20-fold faster than results obtained in vitro.

Using the values of kinetic parameters presented in Table III, we can estimate the contribution of each of the two pathways for the release of AdoHcy in the overall reaction rate. According to the steady state analysis developed earlier (57), the ratio of rates of the two parallel release pathways for AdoHcy can be represented as follows: V_I/V_II = k₄k₁[S]/k₅[S](k₁[S] + k₄[H]). If [H] = 0, then we obtain the simpler expression: V_I/V_II = k₄/(k₅[S]) = 2.9/[S]. From this expression, it is evident that the first pathway predominates when [S] < 2.9 µM and that both pathways are used approximately equally when [S] = 2.9 µM. The second pathway predominates when [S] > 2.9 µM (and its increase in use is proportional to [S] after it exceeds this concentration).

Thus, although some details of the scheme may vary, Scheme 2 can be regarded as a good representation of the reaction mechanism of T4 Dam methylation of oligonucleotide duplexes. At the present time, however, a unified picture of the reaction mechanism of DNA MTases has not emerged despite the numerous studies done on these enzymes. However, the results of the work presented here are important not only in clarifying our understanding of the T4 Dam reaction mechanism, but they may be relevant to the mechanism of other DNA MTases. Specifically, we showed that although free T4 Dam MTase randomly interacts with substrates AdoMet and DNA, it isomerizes in the ternary complex to a new conformational state that is specifically adapted for catalysis. This new conformer performs sequential methylation cycles according to a strictly ordered mechanism of substrate binding and reaction product release. We suggest that the conflicting data obtained with the HhaI MTase (6, 7) can be explained by assuming that this enzyme also undergoes an isomerization after formation of the ternary complex, analogous to T4 Dam.

Based on isotope-partitioning analysis of the HhaI MTase, Lindstrom et al. (6) concluded that any enzyme-AdoMet complex formed is not functionally active and, thus, cannot be part of the reaction mechanism. This conclusion was consistent with the ordered kinetic mechanism, proposed by Wu and Santi (4), in which DNA binds before productive AdoMet binding occurs. However, when Vilkatis et al. (7) repeated this analysis using a significantly higher concentration of labeled AdoMet, their data suggested that the binary HhaI MTase-AdoMet complex could bind DNA in a productive manner and that the enzyme has a random substrate-binding order mechanism. Furthermore, Lindstrom et al. (6) found that the affinity of HhaI for DNA increased 900-fold in the presence of its cofactor, AdoMet, and that at a concentration of AdoMet as high as 1 mM (>100-fold of K_d AdoMet value) no inhibitory effect on the initial velocity or on the burst magnitude (6) was observed. The above data can be reconciled if the HhaI MTase undergoes the same type of isomerization mechanism that occurs with T4 Dam after formation of a ternary complex. Hence, we propose that the HhaI MTase, initially in catalytically inactive form E, interacts randomly with its substrates AdoMet and DNA to form binary complexes, E-AdoMet and E-DNA. After formation of the ternary complex, HhaI-DNA-AdoMet, isomerization produces a catalytically active complex, F-DNA-AdoMet. This is consistent with the data of Vilkatis et al. (7). However, the F form of HhaI MTase then performs sequential methylation cycles according to a strictly ordered mechanism of substrate binding and reaction product release. But, unlike T4 Dam MTase, the HhaI MTase reaction obeys the strict ordered mechanism, DNAAdoMetAdoHcyDNA^me and binds DNA first. Thus, after the conformational change to the F-form, the enzyme's behavior is consistent with the data of Lindstrom et al. (6). The lack of any inhibitory effect of high AdoMet concentration on the HhaI MTase reaction does not contradict this model. Thus, it would appear that an isomerization step to make MTases catalytically active could be a common feature of these enzymes.

	ACKNOWLEDGEMENTS

We thank L. G. Ovechkina and A. V. Zinoviev for their help in the experiments.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant TW00529 from the Fogarty International Center and United States Public Health Service Grant 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.

^¶ To whom correspondence should be addressed. Tel.: 716-274-8046; Fax: 716-275-2070; E-mail: modDNA@mail.rochester.edu.

Published, JBC Papers in Press, October 30, 2001, DOI 10.1074/jbc.M108864200

	ABBREVIATIONS

The abbreviations used are: MTase, methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; DNA^Me, methylated DNA; hmCyt-DNA, 5-hydroxymethylcytosine-containing T4 gt gt (unglucosylated) DNA; MSC, model selection criterion.

	APPENDIX

Kinetic Equation for Scheme 1

Kinetic equation for Scheme 1 (Fig. 5) is as follows in Equation Scheme 1, where e is the total enzyme concentration.

Kinetic Equation for Scheme 2

Kinetic Equation for Scheme 2 (Fig. 5) is as follows in Equation Scheme 2, where e is the total enzyme concentration.

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