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. 2005 Jul;14(7):1929–1933. doi: 10.1110/ps.051445605

Stereo-selectivity of HIV-1 reverse transcriptase toward isomers of thymidine-5′-O-1-thiotriphosphate

Jessica Radzio 1, Nicolas Sluis-Cremer 1
PMCID: PMC2253364  PMID: 15937285

Abstract

The first pre-steady-state kinetic analysis of the stereo-selective incorporation of Rp- and Sp-isomers of thymidine-5′-O-1-thiotriphosphate (TTPαS) by HIV-1 reverse transcriptase (RT) is reported. Rates of polymerization (kpol), apparent dissociation constants (Kd), and substrate specificities (kpol/Kd) were measured for TTP, Rp-TTPαS, and Sp-TTPαS in the presence of Mg2+, Mn2+, and Co2+. HIV-1 RT exhibits a strong preference to incorporate Sp-TTPαS over Rp-TTPαS in the presence of Mg2+; however, this stereo-selective preference was decreased when Mg2+ was replaced with Mn2+ and Co2+. Furthermore, HIV-1 RT exhibited no phosphorothioate elemental effects for the incorporation of Sp-TTPαS, but large elemental effects were calculated for Rp-TTPαS for each of the metals tested. These results are discussed in relation to our current understanding of the RT active-site structure and the mechanism of DNA synthesis.

Keywords: HIV-1, reverse transcriptase, DNA polymerization, stereo-selectivity, pre-steady-state, elemental effect

HIV-1 reverse transcriptase (RT) catalyzes the conversion of single-stranded viral genomic RNA into double-stranded DNA. RT is a multifunctional enzyme that exhibits both DNA polymerase and ribonuclease H activity. Due to its essential role in HIV replication, RT is a major target for drug development (Parniak and Sluis-Cremer 2000). However, due to the high error rate associated with RT-mediated DNA synthesis, as well as other factors such as viral genomic recombination and the selection forces of the human immune system, HIV infections are characterized by a high degree of viral variation (Drosopoulos et al. 1998). This variation gives the virus an ability to escape therapeutic intervention. To better understand HIV replication and drug development at the molecular level, much research has focused on the kinetic mechanism of HIV-1 RT DNA polymerization (Kati et al. 1992; Reardon 1992; Hsieh et al. 1993), the mechanism by which RT inhibitors interfere with reverse transcription (for review, see Parniak and Sluis-Cremer 2000), the mechanisms responsible for error prone DNA polymerization (Weiss et al. 2004), and also the biochemical mechanisms underlying drug resistance (for a recent review, see Deval et al. 2004).

Phosphorothioate elemental effects, derived from experiments which compare the rates of incorporation of the natural dNTP versus dNTPαS, have frequently been used as a diagnostic for determining whether the chemical step of polymerization reactions is rate-limiting (Mizrahi et al. 1985; Kuchta et al. 1987; Patel et al. 1991; Wong et al. 1991). For HIV-1 RT, kinetic studies have demonstrated the absence of a metal effect under normal polymerization conditions (Reardon 1992; Hsieh et al. 1993; Ray et al. 2003), suggesting that the rate-limiting step of the reaction preceded chemical catalysis. However, large metal effects (ranging from 3 to 72) have been detected in HIV-1 RT misincorporation reactions (Zinnen et al. 1994), phosphorolytic excision reactions (Ray et al. 2003), and nucleotide incorporation reactions opposite modified DNA adducts (Choi and Guengerich 2004; Zang et al. 2005). In all of these studies it was proposed that the chemistry step, and not the conformational change preceding chemistry, was rate-limiting. dNTPαS is synthesized as a mixture of two isomers: the pro-Sp isomer (Fig. 1B ▶) and the pro-Rp isomer (Fig. 1C ▶). The orientation of the sulfur in the pro-Sp isomer is such that it faces away from the metal binding site in the active site of DNA polymerases (Fig. 1B ▶). In contrast, the orientation of sulfur in the pro-Rp isomer results in its direct interaction with the metal ions (Fig. 1C ▶). For HIV-1 RT, all kinetic studies that determined elemental effects only considered the Sp-isomer, or alternatively mixtures of both isomers. In order to gain detailed information regarding the interactions between the metal ions and nucleotides at the active site of HIV-1 RT, and also to explore the effects of sulfur substitution on nucleotide incorporation, we analyzed the stereo-selective preference of HIV-1 RT for incorporating both the pro-Sp and pro- RP isomers of TTPαS, using the pre-steady-state kinetic approach. This work represents the first use of pre-steady-state kinetics to study the metal ion dependence and dNTPαS stereo-selectivity in HIV-1 RT.

Figure 1.

Figure 1.

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Schematic representation of the chemistry of DNA synthesis facilitated by HIV-1 RT illustrating the binding interactions of TTP (A), Sp-TTPαS (B), and Rp-TTPαS (C) with Mg2+.

Results and Discussion

Pre-steady-state kinetic analyses of TTP and TTPαS incorporation by HIV-1 RT

Pre-steady-state kinetic analyses of TTP and TTPαS incorporation by HIV-1 RT were undertaken to elucidate the detailed interactions of these nucleotide analogs with the RT polymerase active site. In particular, these experiments were used to define the maximum rate of incorporation (kpol), dissociation constant (Kd), and catalytic efficiency of incorporation (kpol/Kd) for each nucleotide, using three different metal ion cofactors (Mg2+, Mn2+, and Co2+). The data for these experiments are provided in Table 1. The pre-steady-state kinetic parameters determined for HIV-1 RT for the incorporation of TTP and Sp-TTPαS were found to be essentially identical, irrespective of the metal ion used in the assay. In contrast, the catalytic efficiency of incorporation of Rp-TTPαS was ~500-fold slower when Mg2+ and Mn2+ were used as metal ions, and ~50-fold slower when Co2+ was used as the metal ion. This result immediately suggests that HIV-1 RT exhibits a strong preference for Sp-TTPαS over Rp-TTPαS.

Table 1.

Kinetic constants for incorporation of TTP and TTPαS by HIV-1 RT

Nucleotide Metal Kd (μM) kpol (s−1) kpol/Kd (s−1. μM−1) kpol/KdTTP/kpol/KdTTPαS
TTP Mg2+ 2.60 ± 1.41 8.90 ± 1.77 1.17 —
Sp-TTPαS Mg2+ 2.88 ± 1.21 6.77 ± 0.98 9.84 × 10−1 1.19
Rp-TTPαS Mg2+ 45.70 ± 4.75 0.12 ± 0.04 2.62 × 10−3 446.56
TTP Mn2+ 3.44 ± 0.60 5.10 ± 0.79 1.48 —
Sp-TTPαS Mn2+ 4.23 ± 1.04 3.22 ± 0.88 7.61 × 10−1 1.944
Rp-TTPαS Mn2+ 27.32 ± 7.45 0.08 ± 0.12 2.93 × 10−3 505.12
TTP Co2+ 21.40 ± 3.44 2.17 ± 0.52 1.01 × 10−1 —
Sp-TTPαS Co2+ 20.21 ± 4.40 2.27 ± 0.74 1.12 × 10−1 0.90
Rp-TTPαS Co2+ 39.44 ± 6.05 0.084 ± 0.026 2.12 × 10−3 47.6
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The Mg2+, Mn2+, and Co2+ concentrations used in this assay were 10 mM, 2 mM, and 1 mM, respectively.

HIV-1 RT stereo-selectivity toward isomers of TTPαS

Stereo-selectivity is defined as (kpol/Kd)Sp/(kpol/Kd)Rp (abbreviated Sp/Rp ratio), and it evaluates the innate ability of HIV-1 RT to incorporate Rp-TTPαS versus Sp-TTPαS. HIV-1 RT exhibits a pronounced stereoselective preference for Sp-TTPαS over Rp-TTPαS when Mg2+ is used as metal ion (Table 2). This stereoselective preference is due to the Sp-isomer exhibiting both an improved affinity (Kd) for the enzyme’s active site as well as an increased rate of nucleotide incorporation (kpol) compared to the Rp-isomer. A significant relaxation in, but not reversal of, stereo-selectivity is observed when Mg2+ is replaced with the more thiophilic metal ions Mn2+ and Co2+. This observed relaxation suggests that the α-phosphate of TTP is directly chelated by the metal ion during catalysis, data consistent with crystallographic analyses of an RT-T/P-Mg2+- dNTP ternary complex (Huang et al. 1998). Studies evaluating the stereo-selective preference of other DNA polymerases have not been widely reported, except for kinetic studies of Escherichia coli Pol I (Burgers and Eckstein 1979), phage T4 DNA polymerase (Romaniuk and Eckstein 1982), and DNA polymerase β (Liu and Tsai 2001). In contrast to HIV-1 RT, the replacement of Mg2+ by Mn2+, Co2+, or Zn2+ in E. coli Pol 1 did not alter the stereo-selectivity of the enzyme, suggesting that the α-phosphate is not chelated by a divalent metal ion but instead is neutralized by a positively charged group from the enzyme (Burgers and Eckstein 1979). In this regard, the differences observed between the E. coli Pol 1 and HIV-1 RT may reflect subtle differences in the active-site structures of the two enzymes.

Table 2.

Substrate stereo-selectivity of HIV-1 RT toward Sp- and Rp-TTPαS and phosphorothioate elemental effects

Substrate stereoselectivity1 Phosphorothioate elemental effect2
KdSp/KdRp kpolSp/kpolRp kpol/KdSp/kpol/KdRp kpolTTP/kpolSpTTPαS kpolTTP/kpolRpTTPαS
Mg2+ 0.06 56.4 375.6 1.3 74.2
Mn2+ 0.15 40.3 259.7 1.6 63.4
Co2+ 0.51 27.0 52.8 1.0 25.8
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1 Substrate stereoselectivity is defined as a ratio of the Sp-TTPαS and Rp-TTPαS kinetic parameters.

2 Metal effect defined as kpolTTP/kpolTTPαS.

Phosphorothioate elemental effects and the kinetic mechanism of HIV-1 RT DNA synthesis

α-Phosphorothioate-substituted dNTPs have been used to infer the rate-limiting step for polymerization reactions (Mizrahi et al. 1985; Kuchta et al. 1987; Patel et al. 1991; Wong et al. 1991). This approach is based on the observation that substitution of oxygen by sulfur in phosphate triesters results in a 30- to 100-fold decrease in the rate of hydrolysis (Benkovic and Schray 1971). Tomodel reactions of phosphate diesters used in DNA and RNA synthesis more closely, Herschlag et al. (1991) examined the effect of thio substitution on the reaction of the diester, methyl 2,4- dinitrophenyl phosphate, with several different nucleophiles. Replacement of oxygen by sulfur in the diester resulted in more modest elemental effects of 4–11. Thus, the detection of an elemental effect in this range (i.e., 4–11) can oftentimes be indicative of a rate-limiting chemistry event in DNA polymerization reactions.

The elemental effects exhibited by HIV-1 RT toward Rp-and Sp-TTPαS are also reported in Table 2. Consistent with previously published data (Reardon 1992; Hsieh et al. 1993; Ray et al. 2003), elemental effects were not observed for Sp-TTPαS for each of the metal ions tested. However, large elemental effects were observed for Rp-TTPαS. The observed elemental effect for Rp-TTPαS decreased as Mg2+ was replaced with Mn2+ or Co2+. This observed decrease in elemental effect is consistent with the notion that Mn2+ and Co2+ interact better with sulfur than does Mg2+. This result also provides direct kinetic evidence that the Rp-oxygen of the α-phosphate of the incoming dNTP directly interacts with the bound metal ion.

Previous kinetic studies of HIV-1 RT, which observed an RT-T/P-dNTP intermediate by pulse-quench/pulse-chase experiments and the absence of an elemental effect for Sp-TTPαS (Reardon 1992; Hsieh et al. 1993; Ray et al. 2003), suggested that a slow conformational change that precedes chemistry is the rate-limiting step of HIV-1 RT nucleotide incorporation reactions. Does our observation of a large elemental effect for Rp-TTPαS conflict with this interpretation? Elemental effects substantially greater than 11 were observed in other DNA polymerase enzymes (Wong et al. 1991; Polesky et al. 1992). However, the origin of these large elemental effects (including the elemental effect observed for Rp-TTPαS in the present study) may be steric in origin. Sulfur, in addition to being less electronegative than oxygen, also exhibits a larger atomic radius (1.0 Å vs. 0.6 Å for oxygen), and the sulfur-phosphorous bond is longer than the oxygen-phosphorous bond (1.94 Å vs. 1.51 Å). Thus, in an active site where space may be limited, the increased size of the phosphorous-sulfur group could be a major influence on the rate of the reaction. If so, the elemental effect might not exclusively be a diagnostic for the chemical step of the reaction. Alternatively, if the large elemental effect observed for Rp-TTPαS is indeed a “true” elemental effect, this result might suggest that the chemistry step in the HIV-1 RT DNA synthesis reaction may be rate-limiting, or at least partially rate-limiting (i.e., the rate constant for the conformational step that precedes chemistry may be sufficiently slow to mask the slow rate of incorporation of Sp-TTPαS but not that of Rp-TTPαS). In this regard, it was recently proposed that the rate of chemistry in RT-mediated nucleotide addition reactions is in the order of 10–28 sec−1 and that kpol values below this may reflect the rate of the chemical reaction, while for faster chemical reactions the conformational change becomes rate-limiting (Deval et al. 2005). Clearly this finding supports our hypothesis that under certain circumstances the chemical step is rate-limiting in HIV-1 RT DNA synthesis reactions. However, this needs to be validated by using other kinetic techniques, such as stopped-flow fluorescence analyses (Dunlap and Tsai 2002; Purohit et al. 2003), to probe the conformational and chemistry steps involved in RT DNA synthesis.

As described in the Introduction, several HIV-1 RT kinetic studies have used the phosphorothioate elemental effect as the sole diagnostic tool for probing the rate-limiting step of the polymerization reaction. There is already some controversy in the literature as to the validity of exclusively using this approach to probe the rate-limiting steps (Joyce and Benkovic 2004), and our studies clearly highlight this fact by showing that in HIV-1 RT the observed elemental effect is very sensitive to the configuration of sulfur at the active site. Thus researchers should be wary of drawing conclusions from kinetic data that evaluated elemental effects using only Sp-dNTPαS or studies that did not combine elemental effects with other kinetic techniques such as pulse-quench/pulse-chase or stopped-flow fluorescence.

Materials and methods

Reagents

Wild-type (wt) heterodimeric p66/p51 HIV-1 RT was overexpressed as N-terminal hexahistidine fusion protein and purified to homogeneity as described (LeGrice et al. 1995). Enzyme concentration was determined spectrophotometrically at 280 nm using an extinction co-efficient (ɛ280) of 260 450 M−1 cm−1. The Rp- and Sp-isomers of TTPαS were purchased from BioLog (distributed by Alexis). The purity of the isomers is greater than 99% as judged by high-performance liquid chromatography (BioLog). TTP was purchased from Roche Diagnostics. DNA oligonucleotides were synthesized by Integrated DNA Technologies. All other reagents were of the highest quality available and were used without further purification.

Pre-steady-state kinetics of single-nucleotide incorporation

Pre-steady-state DNA polymerization reactions were carried out using a 19-nucleotide DNA primer (5′-GTCCCTGTTC GGGCGCCAC-3′) annealed to a 45-nucleotide DNA template (5′-TAGTCAGAATGGAAAATCTCTAGCAGTG GCGCCCGAACAGGGACA-3′). The DNA primer was 5′-radiolabeled with [γ-32P]-ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (Fisher Scientific) as described in the manufacturer’s protocol, and were then subjected to further purification using denaturing polyacrylamide gel electrophoresis using 7 M urea-14% polyacrylamide gels. The 5′-32P-labeled DNA primer was then annealed to the DNA template by adding a 1:1.5 molar ratio of primer to template at 90°C and allowing the mixture to slowly cool to ambient room temperature.

Rapid quench experiments were carried out using a Kintek RQF-3 instrument (Kintek). In all experiments described, 400 nM RT and 40 nM of the DNA template/primer (T/P) were pre-incubated in reaction buffer (50 mM Tris-HCl [pH 7.5], 50 mM KCl) prior to mixing with an equivalent volume of nucleotide in the same reaction buffer containing a specific concentration of divalent metal ion cofactor. Reactions were terminated at times ranging from 10 msec to 30 min by quenching with 0.5 M EDTA (pH 8.0). Quenched samples were mixed with an equal volume of gel loading buffer (98% deionized formamide, 10 mM EDTA, and 1 mg/mL each of bromophenol blue and xylene cyanol), denatured at 85°C for 5 min, and the products were separated from the substrates on a 7M urea-14% polyacrylamide gel. The disappearance of substrate (19mer) and the formation of product (20mer) were analyzed using a Bio-Rad GS525 Molecular Imager (Bio-Rad Laboratories).

Data analysis

Data obtained from the kinetic assays were fitted by nonlinear regression using Sigma Plot software (Jandel Scientific) with the appropriate equations. To determine the maximum rate of nucleotide incorporation (kpol) and the dissociation constant (Kd) for each nucleotide, the resulting data were fit to a single exponential expression: [T/P+1]=A(1−e−kobst). Kd and kpol values were calculated by fitting the observed single rate constants (kobs) obtained at different concentrations of dNTP to the hyperbolic expression kobs=kpol[dNTP]/(Kd+[dNTP]), where Kd is the equilibrium dissociation constant for the interaction of dNTP with the RT-T/P complex and kpol is the maximum first-order rate constant for dNMP incorporation.

Acknowledgments

This research was supported by NIH grant GM068406-01 to N.S.-C.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051445605.

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