3.7.1. Interaction of M.SsoII with TAMRA-Labelled DNA Containing the Methylation Site in the Presence of AdoHcy
Figure 6 depicts the kinetic traces obtained by detecting changes in TAMRA anisotropy for the M.SsoII interaction with
60met-mC or
60met in the presence of AdoHcy. Initially, an increase in TAMRA anisotropy occurred during the time interval 0 ms to 20 ms in both kinetic series. The anisotropy then showed a slow decrease. The increase likely reflected the formation of an initial enzyme–substrate complex. It was hypothesised that the subsequent decrease in the anisotropy corresponded to isomerisation of the initial complex to produce the second DNA–protein complex. The substrate underwent conformational adjustment during this process. Such double-stage interaction was described using a kinetic scheme containing two reversible steps, which was identical to
Scheme 2.
Fitting the empirical data in DynaFit (
Figure 6) according to this scheme provided the rate constants of the M.SsoII interaction with each duplex with the methylation site (
60met-mC or
60met) in the presence of AdoHcy (
Table 4). The values of the corresponding rate constants of the initial M.SsoII complex formation with
60met-mC or
60met (
k1,
k−1) are comparable. These data revealed that the number of unmethylated Cyt within the methylation site has no significant influence on the initial binding. On the other hand, the number of unmethylated Cyt has an influence on the subsequent isomerisation of the enzyme–substrate complex because the values of the corresponding rate constants
k2* and
k−2* differ for
60met-mC and
60met.
The kinetic traces obtained during the M.SsoII interaction with the methylation site (
60met-mC or
60met) in the presence of AdoHcy, with detection of changes in TAMRA fluorescence intensity are shown in
Figure 7. An increase in the fluorescence intensity occurred in the kinetic series for both duplexes. The initial increase in the time interval 0 ms to 20 ms likely reflected formation of an initial enzyme–substrate complex. The subsequent increase in the fluorescence intensity probably corresponded to the isomerisation of the initial complex.
Fitting the empirical data (
Figure 7A and
Table 4) yielded a four-stage kinetic scheme for the M.SsoII interaction with substrate
60met-mC in the presence of AdoHcy (
Scheme 3).
This result suggested that the formation of the initial enzyme–substrate complex was followed by three isomerisation steps. Thus, the analysis of changes in TAMRA fluorescence intensity allowed us to uncover two additional steps of enzyme–substrate complex isomerisation that were not observed during the monitoring of anisotropy changes. When analysing the fluorescence kinetic traces of M.SsoII interaction with
60met-mC (
Figure 7A), we assumed the kinetic constants of the initial enzyme–substrate complex formation (
k1,
k−1) to be equal to the corresponding rate constants obtained during fitting of the kinetic traces of anisotropy (
Figure 6A). The negligible amplitude of the fluorescence intensity changes in the time interval 0 ms to 20 ms did not allow us to fit these rate constants directly from the fluorescence kinetic traces (
Figure 7A).
The changes in fluorescence intensity at the time points beyond 20 ms were less pronounced in the case of substrate
60met (
Figure 7B) than those for substrate
60met-mC. The kinetic traces of M.SsoII interaction with
60met allowed us to fit only the rate constants of the first reversible step, corresponding to the initial complex formation (
Table 4).
3.7.2. Interaction of M.SsoII with TAMRA-Labelled DNA Containing the Methylation Site in the Presence of AdoMet
M.SsoII interaction with the methylation site in the presence of AdoMet leads to formation of a catalytically competent DNA–protein complex and to subsequent methylation of the target cytosine residues. To monitor this process, the conformational dynamics of TAMRA-labelled DNA 60met-mC or 60met upon its interaction with M.SsoII in the presence of AdoMet was analysed via detection of changes in TAMRA fluorescence intensity and anisotropy.
The kinetic traces obtained for the changes in TAMRA fluorescence intensity are presented in
Figure 8. In the case of substrate
60met-mC (
Figure 8A), the initial multiphase increase in the fluorescence intensity lasted for 4–5 s. This increase likely corresponded to formation of an initial enzyme–substrate complex and its isomerisation. After that, the fluorescence intensity was decreasing (
Figure 8A). Kinetic curves of M.SsoII interaction with
60met-mC in the presence of AdoHcy lacked such a decrease in fluorescence intensity (
Figure 7A). According to these data, we can say that the fluorescence intensity decrease in
Figure 8A corresponds to formation of the catalytically competent complex leading to methylation of Cyt in the substrate. At the same time, TAMRA fluorescence intensity was still increasing at the time points beyond 4–5 s in the kinetic traces during M.SsoII interaction with
60met-mC in the presence of the cofactor analogue (
Figure 7A). This increase corresponded to isomerisation of the enzyme–substrate complex. On the basis of these data, we believe that the enzyme ‘is trying’ to carry out the methylation reaction changing its conformation even under the conditions that prevent substrate methylation, i.e., in the presence of AdoHcy.
The interaction of M.SsoII with
60met in the presence of AdoMet was accompanied by an initial increase in TAMRA fluorescence intensity for 2–3 s likely corresponding to formation of an initial enzyme–substrate complex and its isomerisation (
Figure 8B). The fluorescence intensity then showed a decrease lasting from 2–3 s to 20–30 s. This decrease was consistent with the fluorescence intensity decrease observed in the kinetic traces of
60met-mC during its interaction with M.SsoII at the time points beyond 4–5 s (
Figure 8A). Given these data, the fluorescence intensity decrease from 2–3 s to 20–30 s (
Figure 8B) most likely corresponds to formation of the catalytically competent complex leading to methylation of one Cyt in substrate
60met. The level of fluorescence intensity did not return to its initial value, indicating that M.SsoII did not release the monomethylated substrate. TAMRA fluorescence intensity then showed an increase, and the traces entered a plateau phase (
Figure 8B). The fluorescence intensity increase at the time points after 20–30 s likely corresponds to formation of the catalytically competent complex leading to methylation of the other Cyt. Final levels of TAMRA fluorescence intensity did not return to their initial values in both kinetic series (
Figure 8). This finding indicated that the terminal dissociation step was not reflected in the fluorescence kinetic curves of the M.SsoII interaction with the methylation site in the presence of AdoMet. Thus, the complexes of the enzyme with monomethylated or dimethylated substrates are rather stable.
The M.SsoII interactions with substrate
60met-mC in the presence of AdoMet (
Figure 8A) were described by means of a kinetic scheme containing four steps (
Scheme 4). This scheme included a step of formation of the initial enzyme–substrate complex, and two steps of its isomerisation, followed by a step of formation of the catalytically competent complex leading to the cytosine residue methylation.
We were not able to differentiate between the formation of the catalytically competent complex and the methylation reaction of the cytosine residue. Such step (here and further) was fitted to a reversible equilibrium; therefore, the formation of the catalytically competent complex limits the rate of this step. The methyl transfer reaction itself is irreversible (see
Section 4.1.2. for the explanation), but we could not determine the value of its rate constant.
Fitting the fluorescence traces of
60met during its interaction with M.SsoII in the presence of AdoMet (
Figure 8B) also yielded a kinetic scheme containing four steps (
Scheme 5). In this case, the step of formation of the initial enzyme–substrate complex was followed by one step of its isomerisation, a step of formation of the catalytically competent complex leading to methylation of the ‘first’ cytosine residue and a step of formation of the catalytically competent complex leading to methylation of the ‘second’ cytosine residue.
Thus, the kinetic traces presented in
Figure 8B did not allow us to distinguish the two steps of isomerisation of the enzyme–substrate complex. Fitting of the fluorescence traces provided the rate constants of interaction of M.SsoII with
60met-mC or
60met in the presence of AdoMet (
Table 4). Rate constants
k2* and
k−2* are not the absolute kinetic parameters but rather effective ones describing the joint process of isomerisation. Probably for this reason, the kinetic constant
k−2* was determined with a large error (
Table 4).
Figure 9 represents the kinetic traces obtained during the M.SsoII interaction with
60met-mC or
60met in the presence of AdoMet, with detection of TAMRA anisotropy changes. Initially, an increase in TAMRA anisotropy occurred during the time interval 0 ms to 20 ms in both kinetic series. This increase likely corresponded to formation of the initial enzyme–substrate complex.
In the case of substrate
60met-mC (
Figure 9A), the anisotropy then showed a decrease lasting up to 4–5 s, which likely reflected isomerisation of the initial complex. TAMRA anisotropy then was increased, reaching a plateau (
Figure 9A). This anisotropy increase coincided with the fluorescence intensity decrease at the time points beyond 4–5 s in the kinetic traces presented in
Figure 8A. Besides, kinetic traces of the M.SsoII interaction with
60met-mC in the presence of the cofactor analogue lacked such an increase in anisotropy (
Figure 6A). Due to these findings, the anisotropy increase at time points >4–5 s in
Figure 9A most likely corresponded to formation of the catalytically competent complex leading to methylation of the cytosine residue of
60met-mC.
In the case of substrate
60met, the initial increase in anisotropy was followed by its decrease lasting up to 20–30 s (
Figure 9B). This anisotropy decrease coincided with the TAMRA fluorescence intensity increase and a subsequent decrease (
Figure 8B) corresponding to the enzyme–substrate complex isomerisation and catalytically competent complex formation that precedes the methylation of one cytosine residue. At the time points after 20–30 s, TAMRA anisotropy showed an increase, reaching a plateau (
Figure 9B), which coincided with the rise of fluorescence intensity in
Figure 8B. Thus, the final increase in anisotropy in
Figure 9B most likely corresponded to formation of the catalytically competent complex leading to methylation of the ‘second’ cytosine residue in
60met.
After reaching the plateau phases, TAMRA anisotropy values did not return to their initial values in the kinetic series for both
60met-mC and
60met duplexes (
Figure 9). This result indicated that we did not register the step of DNA product dissociation from its complex with the enzyme in the TAMRA anisotropy traces of
60met-mC or
60met during their interaction with M.SsoII in the presence of AdoMet. These data and the data on changes in TAMRA fluorescence intensity revealed that M.SsoII remained in the stable complex with the DNA product of the enzymatic reaction (monomethylated as well as dimethylated).
The anisotropy traces of
60met-mC during its interaction with M.SsoII in the presence of AdoMet (
Figure 9A) were fitted to a kinetic scheme containing four steps (
Scheme 4). A step of initial enzyme–substrate complex formation was followed by two steps of isomerisation of the complex and a step of formation of the catalytically competent complex leading to the cytosine residue methylation as well as in the case of the TAMRA fluorescence traces (
Figure 8A).
At the same time, fitting the anisotropy traces of
60met upon its interaction with M.SsoII in the presence of AdoMet (
Figure 9B) yielded a kinetic scheme containing three steps (
Scheme 6). The first stage represented formation of the initial enzyme–substrate complex; the second one corresponded to isomerisation of this complex and methylation of one cytosine residue, whereas the third one represented formation of the catalytically competent complex leading to methylation of the other cytosine residue. One can see that the TAMRA anisotropy traces presented in
Figure 9B did not allow us to divide the steps of isomerisation of the initial enzyme–substrate complex and formation of the catalytically competent complex leading to methylation of the ‘first’ cytosine residue. Thus, kinetic parameters
k2** and
k−2** are not absolute but rather effective ones describing the joint process of isomerisation and methylation of the ‘first’ cytosine residue. Although TAMRA has been shown to be rotationally coupled to the DNA motion [
69], TAMRA anisotropy traces of
60met (
Figure 9B) clearly showed that some conformational transitions in the enzyme–substrate complex were not accompanied by TAMRA anisotropy changes. Measuring the changes in TAMRA fluorescence intensity allowed us to register an additional step (which was invisible in the anisotropy traces) in the process of M.SsoII interaction with
60met in the presence of AdoMet. The fluorescence intensity increase corresponding to this additional step is probably due to alterations in the TAMRA environment induced by the rearrangement in the enzyme globule. This rearrangement likely leads to formation of the enzyme conformation that shields TAMRA from the solution, because it is known that a decrease in TAMRA-surrounding polarity significantly increases the fluorescence intensity of a TAMRA–DNA adduct, while the fluorescence anisotropy does not change meaningfully [
70].
Fitting the anisotropy traces provided the rate constant values of the M.SsoII interaction with
60met-mC or
60met in the presence of AdoMet (
Table 4).
Taking into account the strong noise observed in the kinetic curves in the initial time slot (
Figure 8 and
Figure 9), we can say that the values of the corresponding rate constants for formation of the initial M.SsoII complex (
k1,
k−1) with
60met-mC or
60met in the presence of AdoMet closely approximate each other (
Table 4). Thus, the number of unmethylated Cyt bases within the methylation site barely influences the initial enzyme–substrate binding in the presence of AdoMet. All kinetic parameters are discussed below in detail (see the
Section 4).