From the
Human immunodeficiency virus type-1 (HIV-1) reverse
transcriptase (RT) catalyzes DNA synthesis by an ordered sequential
mechanism. After template-primer (T
As expected from earlier
work, the time course of AZTMP incorporation on
poly(rA)-oligo(dT)
Human immunodeficiency virus type-1 (HIV-1),
RT follows an
ordered sequential mechanism for the DNA synthesis reaction pathway.
The deoxynucleoside triphosphate to be incorporated onto the primer
(3`-OH) binds to a complex of template-primer (T
The present report
confirms and extends these earlier findings. We have examined
inhibition of HIV-1 RT by AZTTP during processive dTTP incorporation,
as well as during single-nucleotide incorporation of
2`,3`-dideoxythymidine triphosphate (ddTTP). During processive
nucleotide incorporation, the RT
Heterodimeric recombinant
HXB2R HIV-1 RT (p66/p51) was expressed in Escherichia coli and
purified as described previously (Becerra et al., 1991).
E. coli expressed NY5 recombinant HIV-1 p66/p51 was from
Genetics Institute (Cambridge, MA). A p66 mutant where the leucine at
position 289 was altered to lysine (L289K) was kindly provided by Dr.
R. Goel (Goel et al., 1993). Enzyme concentrations were
determined from protein determinations (Bradford, 1976) that had been
calibrated by amino acid analysis. 5`-[
Reactions
were carried out as described above with 5`-
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Pyrophosphate release from E
When incorporation
is limited to a single nucleotide, the equilibrium dissociation
constant ( k
On-line formulae not verified for accuracy
Data were fitted to appropriate equations by nonlinear least-squares
methods. Inhibition of apparent velocity ( i.e. k
On-line formulae not verified for accuracy
Time course simulations were generated
using HopKINSIM, a Macintosh version of the kinetic simulation program
KINSIM (Barshop et al., 1983) written by D. Wachsstock.
Further details are given in the legend of Fig. 5.
Kedar et
al. (1990) found that the K
For a nucleotide analogue
substrate inhibitor, such as AZTTP, K
The chain-terminating
deoxynucleoside 5`-triphosphates, ddTTP and AZTTP, were incorporated
into a homopolymer of oligo(dT)
The
results suggest that dissociation of E
In , T
For the model depicted in , the
apparent dissociation rate constant determined by a challenge assay, or
k
Ma et al. (1992) reported that the
inhibition constant, K
The
inhibitory potency of AZTTP as measured by the
K
The K
In summary, this study indicates a lack of a simple
correlation between the T
Due to the low amount of incorporation with
ddTTP and AZTTP, the standard error in the fit parameters with these
substrates was as much as 40%. Variation in burst amplitude and
k
We thank Dr. Reeta Goel for providing us with L289K
p66.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P) binds to free enzyme, the
deoxynucleoside triphosphate to be incorporated binds to the RT and
T
P binary complex (RT
). After incorporation of
the bound nucleotide, catalytic cycling is limited either by a
conformational change (for processive synthesis) or release of the
enzyme from the extended T
P (for single-nucleotide
incorporation). To explore cycling through these alternate
rate-limiting steps, we determined kinetic parameters for
single-nucleotide incorporation by HXB2R HIV-1 RT with
chain-terminating nucleotide substrates 3`-azido-3`-deoxythymidine
triphosphate (AZTTP) and dideoxythymidine triphosphate on a
homopolymeric T
P system, poly(rA)-oligo(dT)
.
Inhibition of processive deoxythymidine monophosphate incorporation by
these chain-terminating substrates was also examined. Because AZTTP is
a substrate, its K
should be equivalent
to K
, and since K
for AZTTP should be influenced by the dissociation rate constant
for RT
, we examined the effect of altering
RT
dissociation on AZTTP kinetic parameters. The
dissociation rate constant was modulated by making use of different
T
P substrates, viral sources of RT, and a mutant RT altered at a
residue that perturbs T
P binding.
was biphasic, with a burst followed by
a slower steady-state phase representing k
(0.42
min
) which was similar to the rate constant for
RT
dissociation. Additionally,
K
for AZTTP (110 nM) was lower
than its equilibrium dissociation constant (1200 nM). AZTTP
inhibition ( K
) of
processive dTMP incorporation and incorporation of a single nucleotide
were similar. However, a simple correlation between the RT
dissociation rate constant and
K
was not observed.
These results indicate that a simple ordered model for
single-nucleotide incorporation is inadequate and that different forms
of RT
exist which can limit catalysis. The results
are discussed in the context of a two-step binding reaction for
T
P where the binary RT
complex undergoes an
isomerization before binding of the deoxynucleotide substrate.
(
)
the agent responsible for acquired immunodeficiency
syndrome, encodes a DNA polymerase critical for viral replication. This
enzyme, reverse transcriptase (RT), is an important molecular target
for antiviral therapy. RT catalyzes both RNA and DNA template-dependent
deoxynucleoside 5`-triphosphate (dNTP) incorporation. For example,
3`-azido-3`-deoxythymidine (AZT), which is converted to the
triphosphate form (AZTTP) by cellular kinases, is able to inhibit viral
replication in vivo and RT in vitro (Mitsuya et
al., 1990). Studies of enzyme-inhibitor interaction with an aim to
facilitate drug design have contributed greatly to the understanding of
the RT-catalyzed mechanism. By measuring specific steps in the
multistep reaction scheme, we can undertake structure-function
evaluation of mutant RTs to eventually identify important structural
determinants for enzyme function and inhibition.
P) and enzyme
(Majumdar et al., 1988). The crystallographic structure of
HIV-1 RT has suggested that amino acid residues, whose mutation confers
AZT resistance to the virus, map to a putative single-stranded
template-binding site (Kohlstaedt et al., 1992). In accord
with this idea, the sensitivity of RT to chain-terminating inhibitors
is dependent on the apparent interaction between these residues and the
single-stranded template (Boyer et al., 1994).
Pre-steady-state kinetic characterization of AZTMP incorporation with
HIV-1 RT has also been published (Reardon, 1992). Potent inhibition by
AZTTP of in vitro reverse transcription is the result of
efficient incorporation of the chain-terminating nucleoside analogue
AZTMP (Reardon and Miller, 1990; Reardon, 1992). The slow catalytic
rate in the presence of AZTTP was suggested to be dissociation of RT
from the RT-template-terminated primer complex (Reardon and Miller,
1990; Reardon, 1992), and subsequent studies confirmed this to be the
case (Kati et al., 1992; Reardon, 1993).
complex
dissociation rate constant is not on the primary reaction pathway,
whereas for single-nucleotide incorporation it is a required step.
Incorporation of a chain-terminating nucleotide analogue effectively
limits RT to a single-nucleotide incorporation and, therefore, its
kinetic constants should be influenced by the dissociation rate
constant for T
P, assuming that the simple ordered kinetic scheme
described above is correct. To test this hypothesis, we have examined
the effect of altering the stability of the RT
complex on the kinetic parameters for AZTTP. The RT
dissociation rate constant was modulated by using different
T
P substrates or by taking advantage of altered enzyme/T
P
interactions observed with RT derived from a different strain of HIV-1,
NY5 (Beard and Wilson, 1993), or a site-directed mutant of HXB2R RT,
L289K (Goel et al., 1993). The kinetic scheme described above
was not adequate to explain our results, and a modified scheme is
proposed.
Materials
Poly(rA), oligo(dT),
oligo(dT)
, oligo(dT)
, ddTTP, and dTTP were
from Pharmacia LKB Biotechnol. [
-
P]dTTP
(3000 Ci/mmol) and [
-
P]ATP (4500 Ci/mmol)
were purchased from DuPont NEN and ICN Biochemicals, respectively.
Modified nucleoside triphosphates,
[5-methyl-
H]AZTTP (14 Ci/mmol), unlabeled AZTTP,
and [5-methyl-
H]ddTTP (2 Ci/mmol) were from
Moravek Biochemicals. T4 polynucleotide kinase was from New England
Biolabs. Dupont Nensorb
20 nucleic acid purification
cartridges were obtained from DuPont. DE81 filter discs of 25-mm
diameter were purchased from Whatman.
P] End-labeling of
Oligonucleotides-Oligo(dT) primers were 5` end-labeled with
[
-
P]ATP and T4 polynucleotide kinase at 37
°C for 1 h. Unincorporated [
-
P]ATP was
removed by passing the reaction mixture through a Nensorb
20 column according to the directions given by the manufacturer.
Annealing of Template-Primer
Poly(rA) was
hybridized to oligo(dT), in a 5:1 or 2:1 nucleotide ratio
of template to primer, by heating to 100 °C for 3-5 min and
slowly cooling to room temperature. Substrates not used immediately
were stored at
80 °C. Hybridization of poly(rA) with
radiolabeled oligo(dT)
or oligo(dT)
was
conducted with a 10:1 nucleotide ratio of template to primer.
Concentration of nucleotides, prepared in 10 mM Tris-HCl, pH
7.4, was determined by their UV absorbance at 260 nm using the
manufacturer's extinction coefficients.
Reverse Transcriptase Assay
DNA synthesis
reactions contained 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl, 100 mM KCl, 1 µM
poly(rA)-oligo(dT)
(expressed as 3`-primer termini), dNTP,
and RT as indicated. Reactions were incubated at 20-25 °C and
stopped by quenching with 167 mM EDTA, pH 8.0. Extended primer
was bound to DE81 filters by absorbing the total reaction mixture on
the filters and drying under an infrared lamp. Unincorporated
nucleoside triphosphates were removed by washing the filters four times
with 300 mM ammonium formate, pH 8.0, and twice with 95%
ethanol. The filter discs were dried, and nucleotide incorporation was
quantified by scintillation counting in RPI Biosafe II mixture.
Incorporation was corrected for background radioactivity by measuring
the amount of apparent incorporation in the absence of enzyme for each
concentration of radioactive substrate.
Specific Activity of
With DE81 filter binding, the counting
efficiency of the H-Labeled Nucleoside
5`-Triphosphate
H-labeled triphosphate form of the
nucleoside was found to be lower than that of the
H-labeled
incorporated terminal monophosphate form (Altman and Lerman, 1970).
Hence, incorporated
H-ddNMP bound to DE81 filters was used
as a measure of specific activity. In order to incorporate all of the
H-labeled nucleoside triphosphate (500 nM),
reactions were carried out with excess RT (780 nM) and
T
P (1 µM) for prolonged times (30 min).
Incorporation of labeled
H-ddNTP was considered to be
complete when reaction mixtures were spotted on DE81 filters and
washing did not remove radioactivity.
Primer Extension Assay
Short DNA oligomers,
oligo(dT)and oligo(dT)
, did not bind to DE81
filter discs efficiently. This resulted in an underestimation of the
product formed. Consequently, kinetic parameters with these
oligonucleotides were determined by extension of 5`-radiolabeled
primers and the products resolved on polyacrylamide gels.
P end-labeled
primers and unlabeled dNTP. DNA synthesis reactions were stopped with
EDTA, mixed with an equal volume of gel-loading buffer (0.03%
xylene-cyanol, 0.03% bromophenol blue, and 90% formamide), boiled for 3
min, and cooled immediately on ice. Products ( n+1) were
separated from free primer ( n) by electrophoresis on 28%
polyacrylamide denaturing sequencing gels. Product formation was
quantified by excising the radioactive bands and scintillation
counting.
Kinetic Analysis
A simple model describing
single-nucleotide incorporation is illustrated in where
E represents reverse transcriptase, TP is the
template-primer, dNTP is the deoxy- or dideoxynucleoside triphosphate,
and T
P
is the template-primer after a
single-nucleotide incorporation.
/k
) for
nucleotide binding to E
can be estimated
from the nucleotide concentration dependence of the pre-steady-state
burst amplitude. Under this condition, addition of dNTP to E,
which is saturated with T
P, results in an exponential burst of
nucleotide incorporation followed by a linear steady-state rate of
T
P
accumulation. The rate of the burst can be
followed by rapid mixing and quenching techniques and is sensitive to
the dissociation constant for dNTP binding. The dissociation constant
for dNTP binding to HIV-1RT (Reardon, 1992, 1993; Kati et al.,
1992; Hsieh et al. , 1993) has been determined using this
method. The amplitude of the burst is also sensitive to the
concentration of dNTP and can be determined by extrapolation of the
linear steady-state rate to the ordinate, where
P
represents the ordinate intercept and is
equivalent to the amplitude of the burst (Wharton and Eisenthal, 1981).
) by AZTTP was fitted to:
Figure 5:
Influence of a post-binding isomerization
of the RT complex on the steady-state rate for
single-nucleotide incorporation. Simulated time courses were generated
from Scheme II which includes an isomerization of the RT
and RT
T
The
dissociation rate constant ( i.e. kP Dissociation Rate Constant
) for
T
P from wild type HXB2R and NY5, as well as a L289K mutant of
HXB2R RT was determined as described before (Beard and Wilson, 1993).
Enzyme was preincubated with poly(rA)-oligo(dT)
for 10 min
before challenging with heparin (zero time). Heparin binds free RT, as
well as RT which has dissociated from T
P. At time intervals after
adding challenge, 10-µl aliquots were removed and mixed with 10
µl of dTTP/Mg
to determine the concentration of
RT remaining bound to T
P. The final reaction conditions were 50
nM RT, 75 nM T
P (expressed as 3`-primer
termini), 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl
, 30 µM
[
-
P]dTTP, and 1 mg/ml heparin. EDTA was
added at a final concentration of 167 mM. When the reaction
mixture included 100 mM KCl, the heparin concentration was
increased to 5 mg/ml to adequately compete with T
P for free RT.
Incorporation of radioactive dTMP was determined by spotting the
quenched reaction mixtures on DE81 filters as described in detail
above.
Biphasic AZTMP Incorporation
The time course for
[H]AZTMP incorporation with a
poly(rA)-oligo(dT)
template-primer system was found to be
biphasic (Fig. 1 A). After a steady-state rate of
nucleotide incorporation was achieved, the velocity was constant for
more than 10 min corresponding to a k
of 0.42
min
(). Since the ordinate intercept
extrapolates above the origin, a rapid burst of product formation is
indicated which is too rapid to be measured by manual mixing and
sampling techniques. The linear portion of the time course, expressed
as k
, was independent of enzyme concentration
(Fig. 1 B), whereas the amount of product formed in the
burst phase was dependent upon the RT concentration
(Fig. 1 C). The steady-state velocity was approximately
40-fold lower than determined for incorporation of dTMP under the same
reaction conditions (). Similar observations of a burst
phase followed by a much slower steady-state rate have been made for
AZTMP incorporation (Reardon and Miller, 1990; Reardon, 1992).
Figure 1:
Time course of
[H]AZTMP incorporation by HIV-1 RT with
poly(rA)-oligo(dT)
. The concentration of
[
H]AZTTP and poly(rA)-oligo(dT)
was
10 and 3 µM, respectively, where the T
P
concentration is expressed as concentration of 3`-primer termini. The
template to primer nucleotide ratio was 2. Other reaction conditions
are as described under ``Experimental Procedures.''
A, incorporation of [
H]AZTTP with 8
(
) or 32 nM RT (
). B, time course of AZTMP
incorporation normalized to product formed/enzyme ( P/E). The
ordinate intercept of this plot corresponds to the amplitude of the
burst phase of incorporation which extrapolated to 0.9 AZTMP
incorporated/enzyme. C, a replot of the ordinate intercepts
from panel A illustrates that the burst amplitude is
proportional to the enzyme concentration.
The
amplitude of the burst should be equivalent to the active enzyme
concentration, as long as the rate of product release is much slower
than the rate of AZTMP incorporation ( i.e. k k
, ). The ratio of AZTMP
incorporated during the burst to the amount of enzyme ( P/E)
was 0.7 ± 0.2 ( ordinate intercept in
Fig. 1B) as determined from several independent
experiments. The total enzyme concentration was determined by amino
acid analysis, and this was confirmed by active-site titration with
T
P (Beard and Wilson, 1993).
Steady-state AZTMP Incorporation
Steady-state
kinetic parameters for AZTTP were determined from the incorporation
following the burst. Incorporation increased in a hyperbolic fashion
with increasing AZTTP concentration. From several independent
determinations, the Kwas 110 nM (). The steady-state rate of
incorporation of [
H]AZTMP was much lower than the
rate of incorporation of dTMP. However, the K
for dTTP is significantly greater than for AZTTP, whereas the
efficiency of incorporation calculated by
k
/K
was not
significantly different for these two nucleotide substrates.
Equilibrium Dissociation Constant of AZTTP
The
binding affinity of deoxynucleoside triphosphate for the polymerase
binary complex with TP has been determined by measuring the
concentration dependence of the pre-steady-state rate of a single dNMP
incorporation (Kuchta et al., 1987; Patel et al., 1991; Reardon, 1992, 1993; Kati et al., 1992; Hsieh
et al., 1993). This approach has the advantage that the rate
constant for the incorporation event is also determined. Since the
amplitude of the burst is also sensitive to the
K
of AZTTP, we determined the AZTTP
concentration dependence of the amplitude of the burst phase to obtain
the binding affinity for AZTTP (see ``Experimental
Procedures''). The AZTTP concentration dependence of the square
root of P/E is shown in Fig. 2. Fitting these data to
Equation 2, the equilibrium dissociation constant for AZTTP was
determined to be 1.2 µM ().
Figure 2:
The AZTTP concentration dependence of the
burst amplitude of AZTMP incorporation. Reaction conditions were as
described under ``Experimental Procedures.'' The
concentration of poly(rA)-oligo(dT)and RT was 3
µM and 16 nM, respectively. The template to
primer nucleotide ratio was 5. The concentration of AZTMP incorporated
in the burst was determined by extrapolating the linear steady-state
rate to zero time. Since the maximum incorporation observed in the
burst was P/E = 0.73, the burst amplitude was
normalized to this accumulation. The dissociation constant ( K)
for AZTTP can be estimated from the apparent dissociation constant
( K, see Equation 2) as described under ``Experimental
Procedures.''
AZTTP Inhibition
To examine the inhibition of
single-nucleotide incorporation by AZTTP, the kinetics of another
chain-terminating nucleotide, ddTTP, was characterized. The time course
for ddTMP incorporation was similar to that with AZTMP incorporation.
Following a burst of ddTMP incorporation, a constant rate of
incorporation was observed, and this linear phase extrapolated to a
point on the ordinate that was dependent on enzyme concentration. Like
AZTMP incorporation, k /K
was similar to that for processive dTMP incorporation
(). The K
for AZTTP, as a
competing ligand for single-nucleotide incorporation using ddTTP as
substrate, was 40 nM (Fig. 3 A). In addition,
for processive dTMP incorporation, the K
for AZTTP was 10 nM (Fig. 3 B,
I). Thus, the Michaelis constants for AZTTP and ddTTP, and
the K
for AZTTP, are much lower than the
corresponding equilibrium dissociation constant
( K
) for binding of the nucleotide to the
binary RT
complex (). This difference
will be discussed below.
Figure 3:
Inhibition of single-nucleotide and
processive incorporation by AZTTP. A, the steady-state rate of
[H]ddTTP incorporation was determined from 5 min
of post-burst incorporation. The concentration of ddTTP and RT were 100
and 16 nM, respectively. B, the steady-state rate of
[
P]dTTP incorporation was determined from 20 min
of incorporation. The concentration of dTTP and RT was 3 µM and 2 nM, respectively. Other reaction conditions are as
described under ``Experimental Procedures.'' Inhibition data
were fitted to Equation 3, and the K values were determined to
be 40 and 10 nM for inhibition of single-nucleotide and
processive-nucleotide incorporation,
respectively.
Template-Primer Binary Complex Dissociation Rate
Constant
The observation of a rapid incorporation of
approximately one enzyme equivalent of chain-terminating nucleotide
followed by a slower linear rate of incorporation indicates that a step
following phosphodiester bond formation is rate determining during the
linear steady-state phase. To determine if the steady-state rate is
governed by the dissociation of the RTbinary
complex, the rate constant was measured with a RT challenge assay
(Beard and Wilson, 1993). The dissociation rate constant for
poly(rA)-oligo(dT)
as T
P with a terminal 3`-OH,
under the experimental conditions of this study, was approximately
equal to k
for AZTMP incorporation (data not
shown). This not only demonstrates that T
P dissociation is the
rate-determining step during steady-state single-nucleotide
incorporation on a RNA template (Reardon, 1993), but indicates that the
dissociation rate constant for a primer is not significantly affected
by the presence of an azido group on the sugar moiety.
for
AZTTP was 3 µM using poly(rA)-oligo(dT)
as a
T
P substrate, whereas Reardon and Miller (1990) found it to be
0.22 µM with a similar T
P. predicts
that the K
for a chain-terminating
deoxynucleoside triphosphate is dependent on the dissociation rate
constant for T
P ( i.e. k
;
). When the dissociation rate constant ( k
= k
) is slow relative to
the rate constant for incorporation ( i.e. k
k
k
), the
K
for deoxynucleoside triphosphate is
given by k
( k
+
k
)/ k
k
and
when nucleotide binding is in rapid equilibrium,
K
=( k
k
)/( k
k
)
= K
( k
/k
)
(). K
is,
therefore, expected to be lower than K
by
a factor of ( k
/k
).
When k
k
k
, there is not accumulation of
E
Figure 4:
Dissociation of HXB2R and NY 5 HIV-1
RT/poly(rA)-oligo(dT)complex. Heterodimeric RT was
preincubated with poly(rA)-oligo(dT)
, and at time zero,
heparin was added to bind free RT. At the indicated times,
dTTP/Mg
was added to determine the amount of
RT
complex remaining. The reaction was allowed to
proceed 10 min before stopping with EDTA. The final reaction mixture
contained 50 nM RT, 75 nM poly(rA)-oligo(dT)
, 30 µM dTTP, 1 mg/ml
heparin, 50 mM Tris-Cl, pH 7.4, and 10 mM MgCl
. Data were fitted to a two exponential model:
ES/ES
=
A
e
Effect of RT
It was shown previously that the
dissociation rate constant ( kDissociation Rate
Constant on AZTMP Incorporation
) for
poly(rA)-oligo(dT)
was about 30-fold faster than for
poly(rA)-oligo(dT)
from the RT
binary
complex (Beard and Wilson, 1993). Since this dissociation rate constant
is the rate-determining step with poly(rA)-oligo(dT)
as
T
P, we examined other T
Ps for single-nucleotide
incorporation with AZTTP to determine if k
will
reflect the dissociation rate constant of the T
P. We studied
various circumstances where the RT
dissociation rate
constant was known to be increased (I), e.g. poly(rA) template annealed with oligo(dT)
,
oligo(dT)
, or oligo(dT)
().
Surprisingly, k
for AZTMP incorporation showed
no significant difference with these T
Ps. This is in contrast to
what is predicted from .
is
expected to be approximately equivalent to K
(, ). Since k
for
the binary complex is different with oligo(dT)
and
oligo(dT)
, the K
for AZTTP
with these T
Ps during a single-nucleotide incorporation should be
influenced by this rate constant ( k
) and,
therefore, is expected to be different. Yet, we found that the
K
was not significantly
different. Thus, there is no simple correlation between the
dissociation rate constant for the RT
binary complex
and AZTTP kinetic parameters. To further evaluate the relationship
between the RT
dissociation rate constant and AZTTP
inhibition kinetics, we examined an alternate substrate,
poly(dA)-oligo(dT)
, as T
P. The dissociation rate
constant with this alternate T
P is very rapid and is unmeasurable
by manual mixing methods (I). We found that the
K
for dTMP incorporation
was 15 µM, a value much higher than the
K
observed with
poly(rA)-oligo(dT)
as T
P (I). Thus, in
this case, a faster RT
dissociation rate constant
qualitatively correlated with a higher
K
as predicted from
. Further, the relationship between dissociation of the
RT
complex and AZTTP inhibition was examined with a
site-directed mutant of RT where the leucine at residue 289 was
replaced with lysine ( i.e. L289K). This region has been
identified as forming a portion of the primer-binding site (Sobol
et al., 1991; Basu et al., 1992) and is important for
dimerization of p66 and p51 (Goel et al., 1993). The
dissociation rate constant was determined to be significantly more
rapid than with wild-type enzyme, whereas the K
for AZTTP against dTMP incorporation was not affected
(I). Again, these results indicate that kinetic parameters
for AZTTP inhibition depend on factors not accounted for by
, and the magnitude of k
for the
RT
complex does not quantitatively correlate with
the magnitude of the K
for AZTTP, as
predicted by .
Two-step Binding Reaction for
Template-Primer
The kinetic model in predicts that
the Kfor AZTTP should be sensitive to
the RT
dissociation rate constant
( k
). This is an extremely important concept for
drug design because it links RT and template-primer interactions with
the apparent potency of an inhibitor that is competitive with dNTP
binding. According to this idea, alterations in enzyme-nucleic acid
interactions would also be expected to alter K
for AZTTP. We found here that there are some circumstances where
increases or decreases in RT
dissociation rate
constant do indeed correlate with increases or decreases in
K
. However, there are
other circumstances where such a correlation clearly does not exist.
Therefore, there is no uniform rule defining the relationship between
RT
dissociation rate constant and AZTTP kinetic
parameters. Hence, the model depicted in is not
sufficient to characterize our results.
annealed to poly(rA) in a
biphasic manner. After rapid incorporation of approximately one AZTMP
residue/enzyme, a slower steady-state incorporation of AZTMP occurred
(Fig. 1). The rapid phase represents phosphodiester bond
formation or a conformational change that may limit this event, whereas
the slower phase represents a subsequent step. On a heteropolymeric RNA
template, this has been demonstrated to be T
P dissociation
( i.e. k
) from the binary complex
(Reardon, 1993). Since poly(rA)-oligo(dT)
binds tightly to
RT, K
< 1 nM (Beard and
Wilson, 1993), and since pyrophosphate binds only weakly,
K
in the µM range (Majumdar
et al., 1988), pyrophosphate would be expected to dissociate
much faster than T
P. Additionally, we have determined that
k
for AZTMP incorporation on a homopolymeric
T
P is similar to the dissociation rate constant for the
RT
complex when the primer is at least 16
nucleotides in length (Tables II and III). The steady-state rate for
AZTMP incorporation under low salt conditions was 0.12
min
, which is similar to RT
k
reported for this condition (Beard and Wilson,
1993). However, when shorter primers are employed, the steady-state
rate of chain-terminating nucleotide incorporation is not similar to
the rapid rate at which they dissociate (Tables II and III).
determined with the challenge assay, under some conditions, is
inherently different from T
P dissociation during catalytic
cycling. Thus, is modified to reflect the existence of
another E
complex (designated
* E
) as shown in .
P binding consists of two steps where
k
and k
are the
association and dissociation rate constants for T
P, and
k
and k
are forward and
reverse unimolecular rate constants for a post-binding isomerization.
Previous kinetic characterization of T
P binding to RT has
identified such a post-binding isomerization of the
E
complex (Beard and Wilson, 1993; Divita
et al., 1993; Kruh et al., 1993). In
contrast to , in this model, the dissociation rate
constant for RT
( k
in
) does not have to be equivalent to k
( k
in , ). The
K
for a chain-terminating nucleotide
depends on the apparent dissociation rate constant during catalytic
cycling ( i.e. k
). This may or may not
be equivalent to the dissociation rate constant determined with a
challenge assay.
during single-nucleotide incorporation, will
depend on the magnitude of the unimolecular rate constants. Therefore,
k
and k
can be rapid,
but if k
k
and
k
, the apparent rate constant describing
release of T
P
from
*E
Equilibrium Binding Affinity for Chain-terminating
Nucleoside Triphosphates
The burst amplitude is dependent on the
concentration of the dideoxynucleotide. The apparent
Kdetermined by a plot of the square root
of the P/E underestimates the true dissociation constant by
k
/k
(Fig. 2). In
other words, the dissociation constant for dNTP is diminished by the
ratio of rate constants for incorporation and T
P dissociation.
This ratio of rate constants is equivalent to the processivity of RT
with dTTP as the deoxynucleoside triphosphate (Beard and Wilson, 1993).
Since the efficiency of incorporation
( k
/K
) of AZTMP is
similar to that for dTMP (), the rate of incorporation of
AZTMP was estimated from that for dTMP under processive conditions.
Under these conditions, incorporation is limited by the incorporation
step ( i.e. k
). If dNTP binding is a rapid
equilibrium step, the K
determined under
processive conditions for dTTP is approximately equal to its
dissociation constant. The K
values estimated for
ddTTP and AZTTP are similar to that for the K
of dTTP determined under processive conditions, where
K
is equivalent to
K
(). This result suggests
that our assumptions are valid, and we note that a rapid kinetic
approach also found that the dissociation constants for these
deoxynucleotides were similar (Reardon, 1992). Influence of the T
P Dissociation Rate Constant (i.e.
Processivity) on Inhibition by AZTTP-Inhibition of RT by
AZTTP is competitive with respect to dNTP, and AZTTP has been shown to
be a substrate for HIV-1 RT leading to chain termination (Matthes
et al., 1987; Kedar et al., 1990; Reardon and Miller,
1990). Since RT is a processive polymerase (processivity =
k
/k
1 where
k
is equivalent to k
for single-nucleotide incorporation), the low inhibition constant
reflects premature termination with accumulation of
RT
, experimentally
determined with homopolymeric poly(rA)-oligo(dT) is an artifact of this
T
P system, which is routinely used in these measurements. In the
present study, to ascertain if inhibition by AZTTP is sensitive to the
character of the polymerization reaction, AZTTP inhibition of single
nucleotide (ddTMP) or multiple nucleotide incorporations (dTMP) was
examined on a homopolymeric template. Inhibition of dTTP incorporation
by AZTTP was strong (Fig. 3 B), and Dixon plots were
linear with AZTTP concentrations giving up to 95% inhibition. In
contrast, Reardon and Miller (1990) observed non-linear Dixon plots,
which they postulated may be due to a change in the dissociation rate
constant for RT
complex (see below). Since AZTTP is
also a substrate, predicts that K
should be equivalent to K
. Reardon
and Miller (1990) reported that the K
for
AZTTP was lower than K
. They suggested
that the difference in K
and
K
may reflect a change in the
dissociation rate constant for T
P depending on the number of
nucleotides incorporated. It has been shown that the dissociation rate
constant for the binary RT-homopolymer T
P complex is dependent on
the length of the primer (Beard and Wilson, 1993) and is reflected by a
decrease in the termination probability with increasing number of dTMP
incorporations when a gel electrophoresis assay is used (Huber et
al., 1989; Majumdar et al., 1988; Reardon et
al., 1991). The K
or
K
for AZTTP, therefore, may reflect the
number of nucleotides incorporated into the primer before incorporation
of AZTMP ( i.e. primer length). We found here, however, that
the K
for AZTTP for processive and
single-nucleotide incorporation was similar (Fig. 3).
Additionally, the K
for
processive dTMP incorporation using different length primers and a
homopolymeric template (rA) is also similar (I).
, therefore, is not an artifact of the
homopolymeric T
P as concluded by Ma et al. (1992), but
is the result of the intrinsic processive nature of RT, as well as the
sensitivity of K
to the apparent
dissociation rate constant for T
P (I). Whereas
K
should be equivalent to its
K
as an alternative substrate,
K
is sensitive to the pathway for
incorporation. In one case, as for single-nucleotide incorporation with
a processive polymerase, dissociation of RT from the terminated primer
is an obligatory step and K
underestimates K
by
k
/k
. In the other case,
where processive polymerization can occur such as with a homopolymeric
T
P, K
is equivalent to
K
(). This latter case could
also be achieved if the dissociation rate constant for T
P from
RT
complex were rapid ( i.e. a distributive
polymerase). The variation in K
for AZTTP
with different T
Ps observed by Ma et al. (1992) could be
explained if the apparent dissociation rate constant for T
P was
more rapid when inhibition of single-nucleotide incorporation was
examined on a heteropolymer template (large
K
) than when inhibition of incorporation
was examined on runs of rA (low K
). This
is consistent with the ability of RT to bind much tighter to
poly(rA)-oligo(dT) than RNA/DNA heteroduplexes (Reardon, 1992; Beard
and Wilson, 1993).
reported here
for AZTTP with poly(rA)-oligo(dT)
and HXB2R HIV-1 RT is
much lower than that reported earlier with poly(rA)-oligo(dT)
and NY5 RT (Kedar et al., 1990). The higher value
obtained with NY5 RT is consistent with the more rapid RT
dissociation rate constant observed with NY5 as compared to HXB2R
RT (Fig. 4). Thus, the RT
dissociation rate
constant is dependent on both the length of the primer and the source
of the RT.
P dissociation rate constant and the
level of inhibition observed with AZTTP that would be predicted by a
simple ordered addition of substrates. The apparent dissociation rate
constant during catalytic cycling can, in some instances, be different
from that determined from a challenge assay suggesting that T
P
binding to RT is a two-step reaction resulting in two forms of
E
( i.e. E
and * E
).
Table:
Summary of kinetic constants for HXB2R
recombinant HIV-1 reverse transcriptase and deoxynucleotide
triphosphate binding
from independent experiments was approximately
70%.
Table: 0p4in
Taken from Kedar et
al. (1990). The T
P was
poly(rA)-oligo(dT)
.
P,
template-primer; dNTP, deoxynucleoside 5`-triphosphate; AZT,
3`-azido-3`-deoxythymidine; AZTTP, 3`-azido-3`-deoxythymidine
5`-triphosphate; ddTTP, 2`,3`-dideoxythymidine 5`-triphosphate; dTTP,
deoxythymidine 5`-triphosphate; dNMP, deoxynucleoside monophosphate;
p66, 66-kDa RT polypeptide; p51, 51-kDa carboxyl terminally processed
p66.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.