For more than a decade, we promoted a model in which the conformational change was thought to be rate-limiting and was followed by a very fast chemistry step and fast pyrophosphate release [9
]. This afforded a simplified scheme where nucleotide incorporation was governed by a single rate-limiting step, defined by kpol
, after equilibrium binding of nucleotide to the open
complex with an apparent dissociation constant, Kd,app
is slow relative to the chemistry step (k3
), then the rate of polymerization (kpol
) is defined by k2
, the apparent dissociation constant (Kd,app
) is 1/K1
, and the specificity constant is determined by kcat
. This model afforded a direct method to quantify specificity by measurement of the concentration dependence of nucleotide incorporation in rapid-quench single turnover kinetic studies [13
]. By monitoring a single incorporation event on the time scale of one reaction turnover, rapid quench methods overcome the serious errors introduced in attempting to measure kcat
for a processive enzyme by steady state methods which usually measure the rate of DNA release from the enzyme. As an aside, it should be pointed out that studies using steady state methods to monitor the kinetics of single nucleotide incorporation on processive enzymes are suspect and should be disregarded. Particularly troublesome are rates reported in percent gel band extension per minute [15
], which are difficult to interpret mechanistically. Although one could, in principle, make accurate measurements in the steady state by working at sufficiently low nucleotide concentrations (nM), that is generally not the case. The specificity constants for correct nucleotides are typically underestimated in steady state measurements by a factor of up to 100 and the effects of mutations or altered substrates are masked. This has led to greatly underestimated values for discrimination [16
] and seriously flawed conclusions [12
Rapid quench single turnover kinetic studies provide the best method to measure the kinetic parameters governing sequential nucleotide incorporation during processive synthesis such that kcat
. However, we now know that the simplifications of that allow the assignment of Kd,app
as the nucleotide dissociation constant are incorrect. Rather, the apparent dissociation constant measured in a single turnover experiment should be regarded as a Km
value, which can be much lower than the true Kd
) for the initial collision complex (EDndNTP
]. This will be explained more clearly below.
The central question that we and others have attempted to address is to determine whether the conformational change or the chemistry step is rate limiting. Initial studies were based upon measuring the thio-elemental effect, relying upon substitution of sulfur for a non-bridging oxygen on the α-phosphate of the incoming dNTP to slow the rate of the chemical reaction. Because the thio-elemental effect was small, it was reasoned that the chemistry step must be fast following a rate-limiting conformational change step [10
]. Other studies, including results showing that dNTP binding appeared to be tighter during incorporation by HIV reverse transcriptase in the presence of a nonnucleoside inhibitor which slowed the rate of chemistry, supported the notion of a conformational change leading to tighter nucleotide binding and preceding chemistry [20
Ming-Daw Tsai and his students were the first to provide evidence indicating that the conformational change was faster than chemistry, based upon studies using a fluorescence signal arising from 2-aminopurine in the template strand [21
]. This method was also used in studies on the Klenow fragment of Pol I [23
]. These studies stimulated our studies in which we placed a fluorophore on the fingers domain of T7 DNA polymerase to monitor changes in enzyme structure (). We confirmed that the conformational change was faster than chemistry. Moreover, comprehensive analysis of the kinetics made us realize that comparing the rates of chemistry and the conformational change was only part of the story. It is equally important to determine the relative rates of chemistry and the reverse
of the conformational change leading to nucleotide release. Specificity is not only determined by the relative magnitudes of k2
, but also by the relative rates of k−2
as described below.
The incorporation of a correct nucleotide by T7 DNA polymerase is governed by the rates shown in [19
]. where 1/K1
= 28 µM, defining the dissociation constant in forming the collision complex. Weak binding is followed by a fast conformational change leading to much tighter binding (K2
= 400), which is then followed by the chemical reaction. All evidence suggests that pyrophosphate release and translocation are fast, as described below, so this simple model accounts for the sequential nucleotide incorporation during processive synthesis and these rate constants can be used to compute the specificity constant, kcat
. For this three-step model, kcat
can be derived as:
Interestingly, because k−2
is small relative to k3
, the rate of the chemical reaction drops out of the expression so that the specificity constant reduces to:
The specificity constant further reduces to kcat
= 24 µsM−1
. This term represents the apparent second order rate constant for substrate binding for a two-step binding reaction with a weak rapid equilibrium followed by a fast isomerization. Thus nucleotide selectivity during correct incorporation is based solely upon the rate at which the substrate binds to the enzyme including the isomerization to the form the tight FDndNTP
complex. Once this complex forms, the rate of chemistry does not affect specificity because chemistry is faster than the rate at which the enzyme opens to release bound substrate. That is, once tightly bound, the substrate is committed to go forward and, therefore, it is the binding steps alone that dictate specificity.
Kinetics of correct nucleotide binding
In contrast the binding and incorporation of a mismatched nucleotide (defined by the identity of the templating base) is governed by very different kinetics as shown in .
The initial binding of the mismatch to the collision complex is about tenfold weaker than for a correct base pair. However, the large selectivity against a mismatch occurs with the isomerization to the GDndNTP state from which the rate of chemistry is reduced 1000-fold, while the rate of release of the bound nucleotide is increased 300-fold, relative to a correct nucleotide. Because chemistry is slow relative to nucleotide release, the binding and isomerization come to equilibrium and, therefore, the specificity constant is determined by the product of two equilibrium constants and the rate of chemistry, kcat/Km = K1K2k3 = 0.0008 µM−1s−1.
According to this analysis, nucleotide selectivity is governed by the kinetic partitioning of the enzyme-bound nucleotide species. That is, the probability that a bound nucleotide is incorporated is given simply by the ratio of k3/(k−2+k3). For a correct nucleotide, k−2 is slow relative to k3, so once a nucleotide is bound, it is incorporated most (99.6%) of the time. In contrast, for a bound mismatch, k3 is greatly reduced and k−2 is greatly increased, so it the mismatch is released most (99.9%) of the time. Thus, the changes in enzyme structure following nucleotide binding govern the fate of the bound nucleotide, and the conformational change plays an essential role in establishing enzyme selectivity as dictated by the kinetic partitioning of the FDndNTP (or GDndNTP) state.
We would like ascribe a certain degree of nucleotide selectivity to each step in the pathway in an attempt to define the free energy contribution of the initial binding, the conformational change, and chemistry to net selectivity [24
]. However, this is not so simple. Discrimination is defined as the ratio of the kcat
values derived for correct and incorrect base pairs:
However, for our current model, the discrimination involves the ratio of different kinetic constants for correct and incorrect base pairs:
From this analysis, we can only analyze the effect of changes in K1
for the free energy contribution to discrimination in binding of nucleotide to the open
This level of discrimination is comparable to that observed for a single hydrogen bond. Therefore, these results suggest that the incoming dNTP forms a base pair with the templating base in the open
E.DNA complex. Although the locations of the incoming dNTP and the templating base in the open
complex are not known, these data rule out models suggesting that the dNTP binds first to the enzyme and is then delivered to the template during the conformational change step. Rather, the base pair must form first.
We can also compute the net free energy contribution for discrimination during catalysis by comparing k3 for correct and incorrect base pairs to derive a ΔΔG value of 4.2 kcal/mol. However, this value does not translate linearly to the net nucleotide discrimination because the value of k3 for correct base pair incorporation cancels from the expression defining specificity. Nonetheless it represents the real changes in transition state stabilization affecting the chemical reaction at the active site in comparing a correct base pair and a mismatch, which are most probably related to misalignment of the reactive groups in the mismatch.
Understanding the role of the conformational change in specificity is complex. Because the conformational change is faster than chemistry, the specificity constant defined by K1k2 is greater than would be realized in a pathway in which the conformational change was omitted and the specificity was determined solely by the product of the contributions due to the initial binding and chemistry (K1k3, derived by using the numbering in but bypassing step 2). It is reasonable to suppose that there are limits during evolution on the extent to which an enzyme can achieve discrimination in the chemistry step alone in that further changes in structure that might increase the rate of chemistry for a correct base may also increase the rate of incorporation of a mismatch. The conformational change step allows a disconnect between the rate constants governing the incorporation of a correct base and those governing the incorporation of a mismatch.
The conformational coupling between enzyme structure and fidelity affords further discrimination by altering the structure in response to whether a correct base pair binds to the open
E.DNA complex. It should also be noted that our data indicate that the mismatch recognition state, GDndNTP
is different from the active, closed
, formed after binding a correct base pair. While a correct base induced a decrease in fluorescence of our reporter group, the binding of a mismatch led to an increase, suggesting that there are at least three states: open
and mismatch recognition
]. The fluorescence data, coupled with the observed slower rate of incorporation, led us to postulate a unique mismatch recognition state in which substrate binding energy is used to misalign catalytic residues and slow the rate of catalysis while promoting nucleotide release. This is in contrast to the binding of a correct base in which substrate binding energy is used to organize the active site, hold the nucleotide tightly, and promote catalysis. This new model defines the real power underlying the role of induced fit in enzyme specificity. While the correct substrate induces a structure to facilitate catalysis, the wrong substrate induces a structure to slow catalysis and promote substrate release. This mechanism for achieving increased selectivity may be universal [25
Relating structure to kinetics
According to our working model, specificity is dictated by the changes in enzyme structure that occur after the nucleotide first binds to the open complex (). Little is known about the structure of the initial open EDndNTP complex or where the dNTP binds. However, the observed difference in binding energy between correct and incorrect base pairs in the EDndNTP complex is comparable to what is expected for hydrogen bonding between base pairs (ΔΔG = 1–2 kcal/mole), suggesting that the dNTP interacts with the template base in the open complex via hydrogen bonds. We propose that the shape of the base pair determines the fate of the weakly bound nucleotide during the conformational change step. A correct base pair induces a change in enzyme structure in which the enzyme closes around the base pair to form a tight, catalytic complex. If a mismatched nucleotide is bound, the enzyme does not close, but rather proceeds to a structure which promotes nucleotide release while reducing the rate of catalysis.
A graphic analogy can be made based upon the tasting and swallowing of food. If the initial taste is good, the mouth closes, and in most instances this represents that step at which a commitment is made to swallow the food. In contrast, if the initial taste is bad, that induces an altered configuration of the mouth which promotes release and inhibits swallowing.
Although our model provides a satisfying thermodynamic description of the role of conformational changes in enzyme specificity and efficiency, there are many questions that remain unanswered. In particular, structures of the empty E-DNA state show more disorder in the vicinity of the active site suggesting a more flexible structure than seen the closed E-DNAdd-dNTP state. In addition, there is no structure that shows the binding of a mismatch as an incoming nucleotide. Our kinetic data suggest that the mismatch recognition state is not a single state but a mixture of states, and that may preclude crystallization. Moreover, we do not know the location of the templating base or how the nucleotide first binds to the open E-DNA complex and it may not be possible to obtain a crystal structure of the open complex with nucleotide bound. We can to infer that the dNTP forms a base pair with the templating base because specificity is seen in the initial EDndNTP complex. Following the initial formation of the open EDndNTP complex, we known nothing about how the initial weak interactions trigger a conformational change to the closed state for a correct base, but trigger the formation of a mismatch-recognition state for an incorrect base. This remains as an area of active investigation.
Pyrophosphate release and translocation
Following the chemistry step, the enzyme must release pyrophosphate and then translocates to allow the binding of the next nucleotide. Very little is known about these steps because all evidence suggests that both reactions are normally much faster than chemistry. Evidence for fast pyrophosphate release and translocation come from two experiments. First, the incorporation of two nucleotides in rapid succession occurs as a simple two-step reaction (A→B→C) without any evidence for a kinetically significant step between the two incorporation events [11
]. Second, direct measurement of the rate of pyrophosphate release in a single turnover experiment showed that the rate of the chemical reaction and the rate of pyrophosphate release were coincident [26
]. Because the data indicate that pyrophosphate release is fast following chemistry, there are also little data to assess the reversibility of the chemical reaction at the active site. In single turnover experiments, the rapid release of pyrophosphate drives the reaction to completion. Measurements of the kinetics of the synthesis of a nucleoside triphosphate after adding pyrophosphate from solution are problematic due to the weak binding of pyrophosphate and the low solubility of Mg-pyrophosphate [11
The incorporation of 8-oxo-dGTP and AZT-triphosphate by the human mitochondrial DNA polymerase provides an exception to the general rule that pyrophosphate release is fast. Analysis of the burst kinetics during incorporation of 8-oxo-dGTP showed that the amplitude of the burst was dependent upon the nucleotide concentration implying that the chemical reaction came to equilibrium at that active site of the enzyme [28
]. However, if pyrophosphate release is fast and largely irreversible, then chemistry cannot come to equilibrium in a single turnover, leading to the suggestion that pyrophosphate release must be slow after the incorporation of 8-oxodGTP. Subsequent analysis of the incorporation of the nucleoside analog AZT revealed the same phenomena, and direct measurement showed that pyrophosphate release was extremely slow following the incorporation of AZT [26
]. The reversible chemistry and slow release of pyrophosphate decreases the specificity constant for the incorporation of AZT and 8-oxo-dGTP. Although the structural and thermodynamic basis for this effect is unknown, the results can be rationalized in terms of the physiological challenges of replicating DNA in the highly oxidative environment of the mitochondria. Perhaps the mitochondrial DNA polymerase has evolved this unique means of discriminating against 8-oxo-dGTP, a major oxidative product that accumulates to high concentrations in the mitochondria. Interestingly, in the evolution of resistant to AZT by HIV reverse transcriptase, it appears as though translocation is disfavored which increases the rate of removal of AZT from the DNA by pyrophosphorolysis [3
The most reasonable model for translocation is based upon a fast diffusion of the DNA between the N- and P-sites. When the DNA is bound in the P-site (product site), pyrophosphate can bind to reverse the chemical reaction by the process of pyrophosphorolysis producing dNTP. When DNA is in the N-site (the post-translocation state), it can bind the next nucleotide leading to primer extension. As the DNA rapidly diffuses between the N and P sites, the binding of dNTP captures the DNA at the N-site and the conformational change then locks the nucleotide and DNA in the closed
state. Alternatively, pyrophosphate can trap the DNA at the P-site. This model was proposed some time ago [12
] and has recently been rediscovered and renamed as a “Brownian ratchet” model [31
]. If the equilibrium constant for translocation favors the P-site, then it will appear kinetically and thermodynamically as if dNTP binding drives translocation. Alternatively, if the equilibrium constant favors the N-site, then the nucleotide simply binds to the enzyme after translocation. In either case, translocation is usually fast and not kinetically significant.