At saturation (1 μm
nicked dsDNA substrate), T4 DNA ligase will execute hundreds to thousands of turnovers with a steady state rate of 0.05–0.16 s−1
. The turnover rate varied only 2–3-fold across the substrates tested, with all combinations of natural, correctly base-paired nucleotides at the nick. This rate compared favorably with the turnover rate reported for the ligation of a heterogeneous substrate with many nicks per molecule during early work on T4 DNA ligase (kcat
). The concentration series run for substrates 1
further allowed for estimation of the Michaelis-Menten constants for our substrate system. Although the limitations of the Michaelis-Menten model did not permit the description of the detailed mechanism of DNA ligase activity, the fits gave estimates of the composite rate constants for the system under turnover conditions. The measured value for the kcat
of 150 ± 50 μm−1
for substrate 1
corresponds to an apparent Km
of 2.5 nm
, also very close to the previously reported value of 1.5 nm
for internal nicks in the heterogeneous substrate (20
). Only modest differences were seen in the kcat
between substrates 1
(<2-fold). Together, these data indicated that T4 DNA ligase largely does not discriminate based on the base sequence at single-stranded breaks under multiple-turnover conditions.
The rate constants measured for single turnover ligation of substrate 1
= 5.3 s−1
= 38 s−1
, compared favorably with those measured under single turnover conditions in recent reports for Chlorella
virus DNA ligase (17
) and human DNA ligase I (5
), suggesting that very similar chemical mechanisms operate in all of these ligases despite the ability of T4 DNA ligase to efficiently ligate blunt end substrates. These results conclusively show that AppDNA is a kinetically competent intermediate in ligation by T4 DNA ligase. However, the rate-limiting step for single turnover of substrate 1
is 5.3 s−1
, whereas the rate of turnover under steady state conditions is ~0.4 s−1
. Similarly, the rate-limiting step in the single turnover of AppDNA appears to be 4.2 s−1
, whereas the rate of turnover under steady state conditions is ~0.6 s−1
. In both cases, the rate of the first turnover is ~10 times faster than the turnover rate. A fast single turnover rate in contrast to a slower multiple turnover rate is indicative of product release being the true rate-limiting step.
Previous work has not considered a postligation, pre-enzyme readenylylation rate-limiting step likely for ligases. Upon ligation, the 5′-phosphate and the flexible nick are structurally lost, destroying the ligase binding site. T4 DNA ligase is known to have only a weak affinity for non-nicked DNA (29
); thus, it might have been expected that the ligase would rapidly disassociate from the generated product. However, the results of the pre-steady state burst analysis clearly demonstrated that the rates measured in the separate steady state and single turnover systems do accurately describe the overall behavior of the enzyme. The pre-steady state result showed that, in fact, the rate-limiting step occurs after ligation and is most likely product release or a post-product release conformational change. Similar pre-steady state results have been reported for DNA ligation by the human DNA ligase IV-XRCC4 complex (40
), but, although the authors concluded that the rate-limiting step must be after Step 3, they identified Step 1 enzyme self-adenylylation as the rate-limiting step. Product release was discounted due to the weak affinity of ligases for non-nicked DNA described above. However, enzyme adenylylation is known to be fast for T4 DNA ligase and was confirmed to occur at a rate compatible with the literature values in our system (supplemental Figs. S2 and S3
). Product release is further indicated as the rate-limiting step by the results of AppDNA ligation. It has been previously proposed that there is an additional rate-limiting step present during the ligation of AppDNA, slowing formation of the active complex and accounting for the often seen slower turnover of the intermediate as compared with the phosphorylated substrate (12
). This additional step must take place before initial binding, meaning both single turnover and steady state rates should be affected. However, in our hands, the rate of single turnover of 1A
was again ~10 times faster than the rate of turnover, indicating that the rate-limiting step under turnover conditions must be occurring after ligation.
Given past reports of a weak affinity for non-nicked DNA by T4 DNA ligase, this result may seem surprising. It must be considered that the weak affinity of ligase for non-nicked DNA is reported from gel shift experiments (KD
), not as a measure of on/off rates (29
). Furthermore, immediately after ligation, the DNA-ligase-AMP complex may be in a very different state from the ligase binding nonspecifically to non-nicked dsDNA; for example, the observed rate may well be the relaxation of the “S-complex” seen by Rossi et al.
) in the interaction of deadenylylated enzyme with nicked DNA. Future studies directly investigating DNA binding and disassociation kinetics will allow elucidation of this mechanism.
In the single turnover studies, the rate constants k2
were determined based on a simulation that did not allow for reverse reactions. Previous reports show that high concentrations of PPi
or AMP are needed to observe the reverse reactions under turnover conditions (4
). It is possible, however, that the reactions may be reversible on a single turnover scale before products have disassociated from the active site. It is the case that the residuals seen in A
can be all but eliminated in a model allowing both k−2
to vary (supplemental Fig. S4A
). The simulation requires a value for k−2
3 times larger than the forward rate of k2
, however, and we consider this to be very unlikely, given that the single turnover reaction of AppDNA indicates that k−2
must be at least 50 times smaller than the forward rate. Allowing k−3
to vary while Step 2 is irreversible allows only a modest improvement in the fit (supplemental Fig. S4B
) and indicates that k−3
must be at least 25 times slower than the forward rate of Step 3. Altogether, the single turnover rates indicated that the forward rates of Steps 2 and 3 are substantially faster than the reverse rates. Additional experiments will be needed to accurately determine k−2
It is evident from the reaction curves in that the rate constants for Step 3 determined from the single turnover conversion of substrate 1 and the adenylylated substrate 1A do not agree. The apparent rate of phosphodiester bond formation is only 4.3 s−1 when reacting AppDNA to ligated product, compared with an apparent rate of 38 s−1 when substrate 1 is used. The simplest model that would allow these two experiments to be reconciled is the case of T4 DNA ligase binding slowly to the AppDNA. However, the single turnover experiments at higher ligase concentrations give confidence that binding is fast because a slow binding substrate should show an increased reaction rate when the ratio of enzyme to substrate is increased. Similarly, the experiments using a higher concentration of substrate show no significant change in the reaction progress curve, giving confidence that 100 nm substrate is truly saturating. The analogous experiments performed with substrate 1 indicate that the measured rates are the true single turnover rates for both substrates.
Two major possibilities exist to explain the rate constant discrepancy. First, it is possible that the binding of AppDNA by the apoenzyme produces an active site that differs from the adenylylated enzyme binding phosphorylated nicks, resulting in a rate for the phosphodiester bond formation (k3
) that is simply slower. The second possibility is that there is an additional reaction step not considered by the model used in the data fitting of this study. The two single turnover experiments could not be adequately reconciled using the model proposed for the ligation of nicks by Chlorella
virus DNA ligase (17
), which adds a reversible conversion of the enzyme intermediate complex EX to an inactive complex (GX). Adding such a step to our model did not allow a single model to simultaneously fit both single turnover reactions. Simulations were also performed adding a two-step binding process with an initial fast binding step followed by a slow conversion to an active complex, similar to the additional rate-limiting step model proposed previously for reaction of AppDNA (12
). If the conformational change step occurred with a rate of ~5 s−1
, both experiments could be simultaneously fit reasonably well, but this model predicts a lag phase in the formation of product from 1A
not observed in the data collected (supplemental Fig. S5A
). Another possibility proposed by the same authors was a rate-limiting conformational change in the AppDNA substrate before binding could occur; this model led to nearly identical fits, including a predicted lag phase, as are shown in supplemental Fig. S5A
. The best fits were obtained with a model allowing substrate to bind reversibly in an inactive form as well as directly forming the catalytically active complex (supplemental Fig. S5B
). If AppDNA is much more likely to initially bind in an inactive complex than the phosphorylated nicked substrate, both experiments can be fit to a single model. The constants for the binding rates of DNA are not well constrained by the data available, however, and further experiments into the kinetics of substrate binding will be required to determine which model most accurately reflects the mechanism of T4 DNA ligase.
It was noted that the measured kcat for reaction of substrate 1 at low concentrations (1–50 nm 1, kcat = 0.4 s−1) is ~3-fold larger than the kobs at 1 μm substrate. This result is suggestive that there is some form of substrate inhibition, such as nonspecific DNA binding, taking place. The lower than expected burst amplitude seen in the pre-steady state experiments may likewise be due to substrate inhibition by nonproductive or nonspecific DNA binding. Preliminary simulations allowing inhibition by reversible, nonspecific binding to the DNA indeed showed that both the drop off in turnover rates as DNA concentration increased and the reduced burst amplitude can be accounted for by this sort of substrate inhibition. It is important to note that substrate inhibition cannot account for the presence of the burst phase itself except in the limiting case where the rate of enzyme disassociation from all non-nicked dsDNA is <0.5 s−1. Because this would imply that release of the ligated product DNA would also take place with a rate of 0.5 s−1, the details of inhibition do not impact the key conclusion that a postligation step must be rate-limiting.
This study represents the first detailed analysis of the kinetic mechanism of nick ligation by T4 DNA ligase. Equations 4 and 5 show the model for the ligation pathway, including all constants determined in this study and those previously reported in the literature (4
). We have determined the turnover rates of multiple singly nicked substrates, including one preadenylylated substrate. Furthermore, both the Michaelis-Menten parameters and the single turnover rates of chemistry for one nick and its preadenylylated counterpart have been determined. Finally, we have demonstrated that chemistry is significantly faster than the steady state turnover rate, and, as evidenced by the pre-steady state burst experiment, the most likely candidate for the rate-limiting step of nick sealing by T4 DNA ligase is product release.