©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Immunodeficiency Virus Type 1 Reverse Transcriptase
3`-AZIDODEOXYTHYMIDINE 5`-TRIPHOSPHATE INHIBITION INDICATES TWO-STEP BINDING FOR TEMPLATE-PRIMER (*)

Madhuri Jaju , William A. Beard , Samuel H. Wilson (§)

From the (1) Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068 and Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (TP) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and TP 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 TP (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 TP 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 Kshould be equivalent to K, and since Kfor AZTTP should be influenced by the dissociation rate constant for RT, we examined the effect of altering RTdissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different TP substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs TP binding.

As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)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 RTdissociation. Additionally, Kfor 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 RTdissociation rate constant and Kwas not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTexist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for TP where the binary RTcomplex undergoes an isomerization before binding of the deoxynucleotide substrate.


INTRODUCTION

Human immunodeficiency virus type-1 (HIV-1),() 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.

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 (TP) 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).

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 RTcomplex 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 TP, 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 RTcomplex on the kinetic parameters for AZTTP. The RTdissociation rate constant was modulated by using different TP substrates or by taking advantage of altered enzyme/TP 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.


EXPERIMENTAL PROCEDURES

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 Nensorb20 nucleic acid purification cartridges were obtained from DuPont. DE81 filter discs of 25-mm diameter were purchased from Whatman.

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`-[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 Nensorb20 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 H-Labeled Nucleoside 5`-Triphosphate

With DE81 filter binding, the counting efficiency of the 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 TP (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.

Reactions were carried out as described above with 5`-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 TPis the template-primer after a single-nucleotide incorporation.

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 /k) for nucleotide binding to Ecan 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 TP, results in an exponential burst of nucleotide incorporation followed by a linear steady-state rate of TPaccumulation. 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 Prepresents the ordinate intercept and is equivalent to the amplitude of the burst (Wharton and Eisenthal, 1981).

  

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) by AZTTP was fitted to:

 

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.


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 RTand RT



TP Dissociation Rate Constant

The dissociation rate constant ( i.e. k) for TP 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 TP. At time intervals after adding challenge, 10-µl aliquots were removed and mixed with 10 µl of dTTP/Mgto determine the concentration of RT remaining bound to TP. The final reaction conditions were 50 nM RT, 75 nM TP (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 TP for free RT. Incorporation of radioactive dTMP was determined by spotting the quenched reaction mixtures on DE81 filters as described in detail above.


RESULTS

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 kof 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 TP 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 TP (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 Kfor dTTP is significantly greater than for AZTTP, whereas the efficiency of incorporation calculated by k /Kwas 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 Kof 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 /Kwas similar to that for processive dTMP incorporation (). The Kfor 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 Kfor AZTTP was 10 nM (Fig. 3 B, I). Thus, the Michaelis constants for AZTTP and ddTTP, and the Kfor AZTTP, are much lower than the corresponding equilibrium dissociation constant ( K) for binding of the nucleotide to the binary RTcomplex (). 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 TP with a terminal 3`-OH, under the experimental conditions of this study, was approximately equal to kfor AZTMP incorporation (data not shown). This not only demonstrates that TP 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.

Kedar et al. (1990) found that the Kfor AZTTP was 3 µM using poly(rA)-oligo(dT)as a TP substrate, whereas Reardon and Miller (1990) found it to be 0.22 µM with a similar TP. predicts that the Kfor a chain-terminating deoxynucleoside triphosphate is dependent on the dissociation rate constant for TP ( 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 Kfor deoxynucleoside triphosphate is given by k( k+ k)/ k kand when nucleotide binding is in rapid equilibrium, K=( k k)/( k k) = K( k /k) (). Kis, therefore, expected to be lower than Kby 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/Mgwas added to determine the amount of RTcomplex 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= Ae



Effect of RTDissociation Rate Constant on AZTMP Incorporation

It was shown previously that the dissociation rate constant ( k) for poly(rA)-oligo(dT)was about 30-fold faster than for poly(rA)-oligo(dT)from the RTbinary complex (Beard and Wilson, 1993). Since this dissociation rate constant is the rate-determining step with poly(rA)-oligo(dT)as TP, we examined other TPs for single-nucleotide incorporation with AZTTP to determine if kwill reflect the dissociation rate constant of the TP. We studied various circumstances where the RTdissociation rate constant was known to be increased (I), e.g. poly(rA) template annealed with oligo(dT), oligo(dT), or oligo(dT)(). Surprisingly, kfor AZTMP incorporation showed no significant difference with these TPs. This is in contrast to what is predicted from .

For a nucleotide analogue substrate inhibitor, such as AZTTP, Kis expected to be approximately equivalent to K(, ). Since kfor the binary complex is different with oligo(dT)and oligo(dT), the Kfor AZTTP with these TPs 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 Kwas not significantly different. Thus, there is no simple correlation between the dissociation rate constant for the RTbinary complex and AZTTP kinetic parameters. To further evaluate the relationship between the RTdissociation rate constant and AZTTP inhibition kinetics, we examined an alternate substrate, poly(dA)-oligo(dT), as TP. The dissociation rate constant with this alternate TP is very rapid and is unmeasurable by manual mixing methods (I). We found that the Kfor dTMP incorporation was 15 µM, a value much higher than the Kobserved with poly(rA)-oligo(dT)as TP (I). Thus, in this case, a faster RTdissociation rate constant qualitatively correlated with a higher Kas predicted from . Further, the relationship between dissociation of the RTcomplex 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 Kfor 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 kfor the RTcomplex does not quantitatively correlate with the magnitude of the Kfor AZTTP, as predicted by .


DISCUSSION

Two-step Binding Reaction for Template-Primer

The kinetic model in predicts that the Kfor AZTTP should be sensitive to the RTdissociation 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 Kfor AZTTP. We found here that there are some circumstances where increases or decreases in RTdissociation 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 RTdissociation rate constant and AZTTP kinetic parameters. Hence, the model depicted in is not sufficient to characterize our results.

The chain-terminating deoxynucleoside 5`-triphosphates, ddTTP and AZTTP, were incorporated into a homopolymer of oligo(dT)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 TP 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, Kin the µM range (Majumdar et al., 1988), pyrophosphate would be expected to dissociate much faster than TP. Additionally, we have determined that kfor AZTMP incorporation on a homopolymeric TP is similar to the dissociation rate constant for the RTcomplex 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 RTkreported 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).

The results suggest that dissociation of Edetermined with the challenge assay, under some conditions, is inherently different from TP dissociation during catalytic cycling. Thus, is modified to reflect the existence of another Ecomplex (designated * E) as shown in .

In , TP binding consists of two steps where kand kare the association and dissociation rate constants for TP, and kand kare forward and reverse unimolecular rate constants for a post-binding isomerization. Previous kinetic characterization of TP binding to RT has identified such a post-binding isomerization of the Ecomplex (Beard and Wilson, 1993; Divita et al., 1993; Kruh et al., 1993). In contrast to , in this model, the dissociation rate constant for RT( kin ) does not have to be equivalent to k( kin , ). The Kfor 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.

For the model depicted in , the apparent dissociation rate constant determined by a challenge assay, or kduring single-nucleotide incorporation, will depend on the magnitude of the unimolecular rate constants. Therefore, kand kcan be rapid, but if k kand k, the apparent rate constant describing release of TPfrom *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 TP 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 Kdetermined under processive conditions for dTTP is approximately equal to its dissociation constant. The Kvalues estimated for ddTTP and AZTTP are similar to that for the Kof dTTP determined under processive conditions, where Kis 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 TP 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 kis equivalent to kfor single-nucleotide incorporation), the low inhibition constant reflects premature termination with accumulation of RT

Ma et al. (1992) reported that the inhibition constant, K, experimentally determined with homopolymeric poly(rA)-oligo(dT) is an artifact of this TP 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 RTcomplex (see below). Since AZTTP is also a substrate, predicts that Kshould be equivalent to K. Reardon and Miller (1990) reported that the Kfor AZTTP was lower than K. They suggested that the difference in Kand Kmay reflect a change in the dissociation rate constant for TP depending on the number of nucleotides incorporated. It has been shown that the dissociation rate constant for the binary RT-homopolymer TP 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 Kor Kfor 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 Kfor AZTTP for processive and single-nucleotide incorporation was similar (Fig. 3). Additionally, the Kfor processive dTMP incorporation using different length primers and a homopolymeric template (rA) is also similar (I).

The inhibitory potency of AZTTP as measured by the K, therefore, is not an artifact of the homopolymeric TP as concluded by Ma et al. (1992), but is the result of the intrinsic processive nature of RT, as well as the sensitivity of Kto the apparent dissociation rate constant for TP (I). Whereas Kshould be equivalent to its Kas an alternative substrate, Kis 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 Kunderestimates Kby k /k. In the other case, where processive polymerization can occur such as with a homopolymeric TP, Kis equivalent to K(). This latter case could also be achieved if the dissociation rate constant for TP from RTcomplex were rapid ( i.e. a distributive polymerase). The variation in Kfor AZTTP with different TPs observed by Ma et al. (1992) could be explained if the apparent dissociation rate constant for TP 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).

The Kreported 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 RTdissociation rate constant observed with NY5 as compared to HXB2R RT (Fig. 4). Thus, the RTdissociation rate constant is dependent on both the length of the primer and the source of the RT.

In summary, this study indicates a lack of a simple correlation between the TP 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 TP binding to RT is a two-step reaction resulting in two forms of E( i.e. Eand * E).

  
Table: K

  
Table: Summary of kinetic constants for HXB2R recombinant HIV-1 reverse transcriptase and deoxynucleotide triphosphate binding

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 kfrom independent experiments was approximately 70%.


  
Table: 0p4in Taken from Kedar et al. (1990). The TP was poly(rA)-oligo(dT).


FOOTNOTES

*
This work was supported in part by a grant from the National Institutes of Health Intramural AIDS Targeted Antiviral Program (to S. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: HIV-1, human immunodeficiency virus type-1; RT, reverse transcriptase; TP, 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.


ACKNOWLEDGEMENTS

We thank Dr. Reeta Goel for providing us with L289K p66.


REFERENCES
  1. Altman, S., and Lerman, L. S. (1970) J. Mol. Biol. 50, 235-261 [Medline] [Order article via Infotrieve]
  2. Barshop, B. A., Wrenn, R. F., and Frieden, C. (1983) Anal. Biochem. 130, 134-145 [Medline] [Order article via Infotrieve]
  3. Basu, A., Ahluwalia, K. K., Basu, S., and Modak, M. J. (1992) Biochemistry 31, 616-623 [Medline] [Order article via Infotrieve]
  4. Beard, W. A., and Wilson, S. H. (1993) Biochemistry 32, 9745-9753 [Medline] [Order article via Infotrieve]
  5. Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., and Wilson, S. H. (1991) Biochemistry 30, 11708-11719
  6. Boyer, P. L., Tantillo, C., Jacobo-Molina, A., Nanni, R. G., Ding, J. P., Arnold, E., and Hughes, S. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4882-4886 [Abstract]
  7. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  8. Divita, G., Müller, B., Immendorfer, U., Gautel, M., Rittinger, K., Restle, T., and Goody, R. S. (1993) Biochemistry 32, 7966-7971 [Medline] [Order article via Infotrieve]
  9. Goel, R., Beard, W. A., Kumar, A., Casas-Finet, J. R., Strub, M.-P., Stahl, S. J., Lewis, M. S., Bebenek, K., Becerra, S. P., Kunkel, T. A., and Wilson, S. H. (1993) Biochemistry 32, 13012-13018 [Medline] [Order article via Infotrieve]
  10. Goody, R. S., Müller, B., and Restle, T. (1991) FEBS Lett. 291, 1-5 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hsieh, J. C., Zinnen, S., and Modrich, P. (1993) J. Biol. Chem. 268, 24607-24613 [Abstract/Free Full Text]
  12. Huber, H. E., McCoy, J. M., Seehra, J. S., and Richardson, C. C. (1989) J. Biol. Chem. 264, 4669-4678 [Abstract/Free Full Text]
  13. Kati, W. M., Johnson, K. A., Jerva, L. F., and Anderson, K. S. (1992) J. Biol. Chem. 267, 25988-25997 [Abstract/Free Full Text]
  14. Kedar, P. S., Abbotts, J., Kovacs, T., Lesiak, K., Torrence, P., and Wilson, S. H. (1990) Biochemistry 29, 3603-3611 [Medline] [Order article via Infotrieve]
  15. Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790 [Medline] [Order article via Infotrieve]
  16. Kruh, M., Urbanke, C., and Grosse, F. (1993) Nucleic Acids Res. 21, 3943-3949 [Abstract]
  17. Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., and Benkovic, S. J. (1987) Biochemistry 26, 8410-8417 [Medline] [Order article via Infotrieve]
  18. Ma, Q.-F., Bathurst, I. C., Barr, P. J., and Kenyon, G. L. (1992) Biochemistry 31, 1375-1379 [Medline] [Order article via Infotrieve]
  19. Majumdar, C., Abbotts, J., Broder, S., and Wilson, S. H. (1988) J. Biol. Chem. 263, 15657-15665 [Abstract/Free Full Text]
  20. Matthes, E., Lehmann, Ch., Scholz, D., von Janta-Lipinski, M., Gaertner, K., Rosenthal, H. A.,and Langen, P. (1987) Biochem. Biophys. Res. Commun. 148, 78-85 [Medline] [Order article via Infotrieve]
  21. Mitsuya, H., Yarchoan, R., and Broder, S. (1990) Science 249, 1533-1544 [Medline] [Order article via Infotrieve]
  22. Müller, B., Restle, T., Reinstein, J., and Goody, R. S. (1991) Biochemistry 30, 3709-3715 [Medline] [Order article via Infotrieve]
  23. Patel, S. S., Wong, I., and Johnson, K. A. (1991) Biochemistry 30, 511-525 [Medline] [Order article via Infotrieve]
  24. Reardon, J. E., and Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307 [Abstract/Free Full Text]
  25. Reardon, J. E., Furfine, E. S., and Cheng, N. (1991) J. Biol. Chem. 266, 14128-14134 [Abstract/Free Full Text]
  26. Reardon, J. E. (1992) Biochemistry 31, 4473-4479 [Medline] [Order article via Infotrieve]
  27. Reardon, J. E. (1993) J. Biol. Chem. 268, 8743-8751 [Abstract/Free Full Text]
  28. Segel, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, John Wiley & Sons, New York
  29. Sobol, R. W., Suhadolnik, R. J., Kumar, A., Lee, B. J., Hatfield, D. L., and Wilson, S. H. (1991) Biochemistry 30, 10623-10631 [Medline] [Order article via Infotrieve]
  30. Wharton, C. W., and Eisenthal, R. (1981) Molecular Enzymology, Blackie, London

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