Human immunodeficiency virus type-1 reverse transcriptase. Contribution of Met-184 to binding of nucleoside 5'-triphosphate.

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the kpvalues of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5'-triphosphate.

kinetic step identified as k p ; the concentration of template-primer is much greater than its dissociation constant for free enzyme; and the kinetic constants for translocation of the enzyme on E⅐TP nϩ1 , binding of dNTP, and dissociation of PP i are large relative to k p . Processive synthesis is defined as DNA synthesis in which multiple nucleotides are incorporated into the primer by the polymerase prior to dissociation of E⅐TP n (k off Ͻ Ͻ k p ) (Scheme 1), where TP n is the template-primer, TP nϩ1 is the template-primer after incorporation of one nucleotide, and dNTP is the nucleoside 5Ј-triphosphate. Under these conditions, K m ϭ K d and k cat ϭ k p . Distributive synthesis occurs when the E⅐TP nϩ1 complex dissociates after each nucleotide addition (Scheme 2). During nonprocessive synthesis, k off Ͼ k p , K m ϭ K d , and k cat ϭ k p . During forced termination of processive synthesis, as is the case with incorporation of a chain-terminating nucleotide analog, k off Ͻ Ͻ k p , K m ϭ k off (K d /k p ), and k cat ϭ k off .
Synthesis of Nucleoside Analog 5Ј-Triphosphates-1-␤-L-FTCTP, BCH189 5Ј-triphosphate, 3TCTP, and 1-␤-L-ddCTP were prepared as described by White et al. (21), except that 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone was substituted as solvent. After the solvent was removed from the reaction mixture by extraction with chloroform, the nucleoside analog 5Ј-triphosphate (0.01 mmol) was purified by ionexchange chromatography. The products were absorbed onto a Pharmacia Mono Q 10/10 anion-exchange column equilibrated in 0 mM ammonium bicarbonate, and the nucleoside analog 5Ј-triphosphate was eluted with a linear gradient of ammonium bicarbonate from 0 to 700 mM. 1-␤-L-FTCTP was eluted at 350 mM ammonium bicarbonate in a single peak, resulting in a 34% yield; -3TCTP and BCH189 5Ј-triphosphate were each eluted at 280 mM ammonium bicarbonate in a single peak, resulting in 21 and 20% yields, respectively; and 1-␤-L-ddCTP was eluted at 210 mM ammonium bicarbonate in a single peak, resulting in a 19% yield. 1 H NMR and 31 P NMR spectra corresponded with those expected for dideoxynucleoside 5Ј-triphosphates. Template-Primers-Templates and primers were annealed by heating a solution of template and primer in a defined ratio at 65°C for 3 min and slowly cooling to room temperature (17). The 22-base oligomer (5Ј-CCC T-GC GGG ATG TGG TAT TCC T-3Ј) was annealed with single-stranded M13mp18HXBRT as follows. 7.75 g of the primer at 25 g/ml was mixed with 155 g of the single-stranded M13mp18HXBRT vector at 1 mg/ml. M13mp18HXBRT is the entire 1.7-kilobase HIV-1 RT coding region inserted into the EcoRI and HindIII sites in the polylinker of M13mp18 (22). The primer is complementary to bases 285-300 in the HIV-1 RT coding region. This primed DNA was designated pM13mp18HXBRT. Poly(rI) and oligo(dC) 19 -24 were annealed in a 1:1 ratio of template to primer with respect to the 5Ј-ends to yield poly(rI)⅐(dC) 19 -24 . The sequences of the defined sequence r44-mer, d23-mer, and d24-mer used for steady-state and presteady-state kinetic studies are as follows: r44-mer, 3Ј-CCC CUA G-GA GAU CUC AGC UGG ACG UCC GUA CGU UCG AAC AGA GG-5Ј; d23-mer, 5Ј-GGG GAT CCT CTA GAG TCG ACC TG-3Ј; and d24-mer, 5Ј-GGG GAT CCT CTA GAG TCG ACC TGC-3Ј. The r44-mer was synthesized using MEGAscript TM as described below. The r44-mer was annealed in a 1:2 ratio with respect to the 5Ј-ends with each d23-mer and d24-mer to yield r44⅐d23-mer and r44⅐d24-mer, respectively. The sequences for the d45-mer template and the d29-mer primer are as follows: d45-mer, 3Ј-CGG AGC-CTC GGC AGG TTG GTT GAG TTC GAG CTA GGT TAC GGC AGG-5Ј; and d29-mer, 5Ј-GCC TCG CAG CCG TCC AAC CAA CTC AAC CT-3Ј. They were annealed in a 1:2 ratio of template to primer with respect to the 5Ј-ends to yield d45⅐d29-mer.
The Ready-To-Go TM T4 polynucleotide kinase kit was used to label the d23-mer with 32 P to yield [ 32 P]d23-mer for rapid-quench experiments. The instructions included by the manufacturer were followed. The specific activity of [␥-32 P]ATP was 6000 Ci/mmol. After the reaction was terminated, [ 32 P]d23-mer was purified by two Bio-Rad Biospin desalting columns (following the instruction provided by the manufacturer). For the rapid-quench experiments, the r44-mer and [ 32 P]d23mer were annealed in a 6:1 ratio with respect to the 5Ј-ends so that Ͼ90% [ 32 P]d23-mer was bound to the r44-mer, yielding r44⅐ [ 32 P]d23-mer.
Synthesis of r44-mer-The r44-mer was synthesized using the MEGAscript TM large-scale T7 transcription kit. The sequences of the template and primer used for synthesis are as follows: T7PRIM, 5Ј-TAA TAC GAC T-CA CTA TAG-3Ј, and T7TEM, 3Ј-ATT ATG CTG AGT GAT ATC CTC T-GT TCG AAC GTA CGG ACG TCC AGC TGA GAT CTC CTA GGG G-5Ј. They were annealed in a 1:1 ratio with respect to the 5Ј-ends to yield T7TEM⅐T7PRIM. The r44-mer was synthesized as described in the instructions provided with the MEGAscript TM kit with modification for synthesis of a short transcript (23). A concentration of 50 g/ml T7TEM⅐T7PRIM was used in the reaction. The reaction was allowed to proceed for 6 h at 37°C, after which the DNA template was removed by adding RNase-free DNase to the solution at 37°C for 15 min. The reaction was terminated with ammonium acetate. The r44-mer was purified by extraction with phenol/chloroform and extraction with chloroform. The solution containing the r44-mer was further purified by three Bio-Rad Biospin columns to remove nucleoside 5Ј-triphosphates. The concentration of the r44-mer was determined from the A 260 and ⑀ 260 ϭ 399,000 M Ϫ1 cm Ϫ1 (24).
Steady-state Kinetic Assays-All steady-state enzyme assays were performed at 37°C as described previously (22). Five aliquots of the reaction solution were removed during the linear steady-state phase of the reaction and spotted on DE81 paper. The paper was then washed with 5% sodium phosphate, dried, and counted in a Packard Tri-Carb 2500CA liquid scintillation counter. Rates of product formation were calculated from these data by linear regression analysis. Steady-state kinetic parameters (K m and k cat ) were determined by nonlinear regression analysis using Enzyme Kinetics (Trinity Software, Campton, NH). Inhibition constants were determined by Dixon plot analysis. Active enzyme concentrations were determined as described by Reardon and Miller (16). Reactions with poly(rA)⅐(dT) 10 and poly(rC)⅐(dG) 12-18 contained 50 mM Tris-HCl (pH 7.8), 5 mM MgCl 2 , and 0.025% Triton X-100 (Buffer A). Reactions with pM13mp18HXBRT contained Buffer A SCHEME 1 SCHEME 2 with 25 mM KCl. Reactions with poly(rI)⅐(dC) 19 -24 contained Buffer A with 2 mM MgCl 2 instead of 5 mM (higher MgCl 2 concentrations caused precipitation of the template-primer). Reactions with r44⅐d23mer and r44⅐d24-mer contained Buffer A with 50 mM KCl. All assays were initiated with enzyme with the exception of those with poly-(rI)⅐(dC) 19 -24 , which were initiated with the template-primer.
Pre-steady-state Kinetic Assays-Pre-steady-state kinetic data were collected as described previously by Reardon (24) using a rapid-mixing apparatus purchased from Kin-Tec (25). Briefly, the enzyme⅐radiolabeled template-primer complex in 50 mM Tris-HCl (pH 7.8), 50 mM KCl, 2.5 mM EDTA, and 0.025% Triton X-100 (Buffer B) was mixed at 25°C with varying concentrations of substrate in Buffer B with 15 mM MgCl 2 . The reactions were quenched with 0.5 M EDTA at selected times between 0.01 and 10 s after initiation of the reaction. Reactions to determine the kinetic parameters of wt RT for dCTP and 1-␤-L-FTCTP contained 200 nM enzyme, 10 nM r44⅐[ 32 P]d23-mer, and substrate at selected concentrations ranging from 1 to 20 M. Reactions to determine the kinetic parameters of M184V RT for 1-␤-L-FTCTP contained 400 nM enzyme, 10 nM r44⅐[ 32 P]d23-mer, and 1-␤-L-FTCTP at selected concentrations ranging from 400 to 3 M. The quenched samples (100 l) were desalted by two Bio-Rad Biospin columns. The desalted samples were lyophilized; resuspended in a solution of 45% formamide, 0.025% xylene cyanol FF, and 0.025% bromphenol blue; heated at 90°C for 5 min; and rapidly cooled on ice. The products were separated by electrophoresis on an 8 M urea, 15% polyacrylamide gel, and the DNA was quantitated with a Molecular Probes PhosphorImager and the software Image-Quant. Equation 1 was fitted to the time courses for product formation, where A is the amplitude of the burst, k p is the observed first-order rate constant, [S] is the nucleoside (analog) 5Ј-triphosphate concentration, and t is time. Five to six concentrations of substrate were used to determine the nucleoside (analog) 5Ј-triphosphate dissociation constant (K d ) and burst rate constant (k p ).

Steady-state Kinetics for Nucleotides-
The K m and k cat values of wt RT, M184V RT, and M184A RT were determined for the natural dNTPs. The k cat values of M184V RT and M184A RT were not significantly different from those of wt RT for RNA-and DNA-directed DNA synthesis, indicating that neither of the mutations affected the rate-limiting step of the reaction (Table I).
The k cat is equal to the k p for RT-catalyzed nucleotide incorporation into the homopolymeric template-primers poly(rI)⅐(dC) 19 -24 , poly(rC)⅐(dG) [12][13][14][15][16][17][18] , and poly(rA)⅐(dT) 10 , which were utilized to determine the kinetic constants for dCTP, dGTP, and dTTP, respectively. The k cat values of wt RT and M184V RT for dATP incorporation for RNA-directed synthesis were significantly smaller than those for dCTP, dGTP, and dTTP incorporation. In this case, RT catalyzed the incorporation of dAMP into the defined sequence template-primer r44⅐d23-mer, where the only nucleotide present was the next encoded nucleotide, dATP; therefore, the enzyme was forced to dissociate from the template-primer after each dAMP incorporation. Under these circumstances of forced termination of processive synthesis, k cat is equal to k off for E⅐TP nϩ1 .
The K m values of M184V RT for the natural dNTPs during either RNA-or DNA-directed DNA synthesis were not different from those of wt RT, suggesting that the mutation to valine did not significantly affect natural substrate binding (Table I). In addition, the K m values of M184A RT for dCTP and dTTP for DNA-directed synthesis were similar to those of wt RT. In contrast to DNA-directed synthesis, the K m values of M184A RT for dCTP and dTTP for RNA-directed synthesis were 14and 70-fold larger than those of wt RT, respectively. Therefore, the K m values of M184A RT were larger than those of wt RT specifically for RNA-directed synthesis. To determine if this difference between RNA-and DNA-directed synthesis found for M184A RT was due to the template-primers used (homopolymeric template-primers were used for RNA-directed synthesis, and the heteropolymeric template-primer pM13mp18HXBRT was used for DNA-directed synthesis), K m values were determined using the template-primers r44⅐d23-mer and d45⅐d29mer under conditions of forced termination in which the first nucleotide to be incorporated into each template-primer was dCTP. For r44⅐d23-mer, the K m value for dCTP of wt RT was 21 nM, and that of M184A RT was 800 nM; the substitution of alanine for Met-184 resulted in a 40-fold larger K m value for RNA-directed synthesis. With d45⅐d24-mer as the templateprimer, the K m value for dCTP of wt RT was 200 nM, and that of M184A RT was 320 nM; substitution of alanine for Met-184 did not significantly affect the K m value for dCTP. Thus, the K m value of M184A RT was significantly larger than of wt RT for RNA-directed synthesis, but not for DNA-directed synthesis.
Steady-state Kinetics for Nucleotide Analogs-The K i values of wt RT and M184V RT for 1-␤-Dand 1-␤-L-nucleotide analogs were determined for both RNA-and DNA-directed DNA synthesis (Table II). The K i values for both 1-␤-D-and 1-␤-L-enantiomers of cytidine, guanosine, and thymidine nucleotide analogs were determined to evaluate enantiomeric selectivity of M184V RT.
The K i values of wt RT for inhibitors of RNA-directed synthesis varied from 3 nM to 2 M. This is not a reflection that, for example, ddGTP (with a K i value of 4.2 nM) binds to RT better than ddCTP (with a K i value of 770 nM). As was noted previously, these differences were due to the fact that different template-primers were used for each nucleotide bases assayed. Therefore, only the K i values determined using the same individual template-primer can be compared meaningfully.
The K i values of M184V RT for ddATP (the active metabolite of dideoxyinosine) and ddCTP for RNA-directed synthesis were   Table III. The K i values of M184A RT for 1-␤-Dand 1-␤-L-ddCTP were increased by 5-and 3-fold relative to wt RT, respectively. The K i values of M184A RT for the thiacytidine analogs were increased 40-, 30-, 30-, and 85-fold relative to wt RT for BCH189 5Ј-triphosphate, 3TCTP, and 1-␤-Dand 1-␤-L-FTCTP, respectively. Because the K m value of M184A RT for dCTP was 14-fold larger than that of wt RT, it is more appropriate to compare differences seen in the K i /K m ratios of wt RT and M184A RT to determine if utilization of the nucleoside analogs has been more significantly compromised than dCTP utilization. When these values were analyzed, no significant differences were observed (Table III). In fact, the K i /K m ratios of M184A RT for 1-␤-D-and 1-␤-L-ddCTP were lower than those found for wt RT. These data suggest the K m values of M184A RT for all nucleotide (analog) substrates, regardless of stereochemistry, have been affected to approximately the same extent by the substitution of Met-184 with alanine.
Pre-steady-state Kinetics for wt RT and M184V RT-The equilibrium dissociation constant (K d ) and the burst rate constant (k p ) of wt RT and M184V RT for dCTP and 1-␤-L-FTCTP were calculated from the concentration dependence of the pseudo first-order rate constant for dNMP incorporation into template-primer (Equation 1). The experimental conditions were such that, after the first round of incorporation of substrate in the burst phase of the reaction, r44⅐[ 32 P]d23-mer was exhausted, and no steady-state rate of substrate incorporation was observed. Fig. 2 shows these data for wt RT and M184V RT with 1-␤-L-FTCTP as the substrate. These data are not shown for dCTP. For wt RT, the dissociation constant for dCTP was 16 a r44 ⅐ d23-mer was used for dATP turnover, poly(l) ⅐ (dC) 18 -24 for dCTP turnover, poly(rC) ⅐ (dG) [12][13][14][15][16][17][18] for dGTP turnover, and poly(rA) ⅐ (dT) 10 for dTTP turnover.
b The template-primer used for all dNTPs for DNA-directed synthesis was pM13mp18HXBRT as described under "Experimental Procedures." c ND, not determined.
Ϯ 5 M, and the burst rate constant was 9 Ϯ 2 s Ϫ1 ; and the dissociation constant for 1-␤-L-FTCTP was 1.7 Ϯ 0.3 M, and the burst rate constant was 0.24 Ϯ 0.02 s Ϫ1 . To determine what effect the mutation at position 184 to valine had on the kinetic constants for dCTP and 1-␤-L-FTCTP, pre-steady-state kinetics were also determined with M184V RT for these substrates. The K d value of M814V RT for dCTP of 40 Ϯ 10 M was slightly higher than the K d value of wt RT, but the mutation had no effect on the burst rate constant (9 Ϯ 2 s Ϫ1 ). The dissociation constant of M184V RT for 1-␤-L-FTCTP was 19-fold larger than that of wt RT; again, the burst rate constant was unaffected (0.18 Ϯ 0.03 s Ϫ1 ). These values are summarized in Table IV, along with the steady-state K m values experimentally determined and the steady-state K m values calculated by using the relationship K m ϭ k off (K d /k p ), where k off is the k cat value determined for dCTP under steady-state assay conditions.

DISCUSSION
The YMDD motif spanning amino acids 183-186 of HIV-1 RT has been reported to comprise part of the putative active site of the enzyme (26,27). A substitution of alanine at position 184 in this highly conserved region significantly compromised the replication rate of the resulting mutant virus, whereas a substitution of valine had no effect (11). The k cat values of both mutant enzymes for dTTP turnover with poly(rA)⅐(dT) 10 and for dCTP turnover with poly(rI)⅐(dC) 19 -24 were similar to those determined for wt RT. HIV-1 RT catalyzes processive synthesis with poly(rA)⅐(dT) 10 as the template-primer and nonprocessive synthesis with poly(rI)⅐(dC) 19 -24 as the template-primer; k cat is equal to k p for both types of synthesis, based on the simplified kinetic model. In addition, k cat values of M184A RT and M184V RT for dTTP and dCTP using M13mp18HXBRT as the template-primer were not different from values determined for wt RT; again, k cat is equal to k p . The k cat values of M184V RT for dATP and dCTP and of M184A RT for dCTP for RNA-directed synthesis were also determined under conditions of forced termination with the template r44-mer and primers d24-mer and d23-mer, respectively; these values were similar to those determined for wt RT. In the case of forced termination of processive synthesis, k cat is equal to k off for E⅐TP nϩ1 . These data indicate that substitution of valine or alanine for Met-184 did not affect k p , k off for E⅐TP nϩ1 , and the processivity of RT (which is determined by the ratio of k p to k off ).
The K m values of M184A RT were larger than those of wt RT for processive synthesis (poly(rA)⅐(dT) 10 ), nonprocessive synthesis (poly(rI)⅐(dC) 19 -24 ), and forced termination of processive synthesis (r44⅐d23-mer). The K m value for substrate during processive and nonprocessive synthesis is equal to the K d value for dNTP binding to E⅐TP n . The K m value for substrate during forced termination of processive synthesis is equal to k off (K d / k p ). Because the values of k off and k p for M184A RT were similar to those of wt RT, the equilibrium binding constant of M184A RT for dNTP has been affected by the substitution at Met-184. No significant effect was observed on the K m values of M184V RT for dNTPs during processive or nonprocessive syn-

values of wt RT, M184V RT, and M184A RT for cytidine analogs
The template-primer used was poly(l) ⅐ (dC) 18 -20 . K i values for wt RT and M184V RT were also reported in Table II.  Table I. thesis or forced termination of processive synthesis. Thus, the catalytic efficiency of M184A RT (k cat /K m ) is less than that of M184V RT and wt RT. Therefore, the slower replication rates of M184A HIV-1 relative to wt HIV-1 and M184V HIV-1 in cell culture are probably due to an increase in the K d value for dNTP binding to E⅐TP mϩ1 , possibly allowing reverse transcription to become rate-limiting in the replication life cycle of M184A HIV-1.
Interestingly, the K m values of M184A RT for dNTPs are larger than those of wt RT only for RNA-directed DNA synthesis. Similarly, the K i values of M184V RT for ddATP and ddCTP (the active metabolites of dideoxyinosine and dideoxycytidine, respectively) were larger (6-and 3-fold, respectively) than those of wt RT only for RNA-directed DNA synthesis. These results indicate that Met-184, in conjunction with the template-primer, contributes to the binding energy for nucleotide substrates such that mutations specifically affect binding of a 1-␤-D-nucleotide to an HIV-1 RT⅐RNA-DNA complex. A DNA-DNA helix usually adopts a B-form configuration free in solution (28,29). However, when HIV-1 RT is co-crystallized with a double-stranded DNA template-primer, the DNA adopts an A-form configuration in the polymerase domain (30). The configuration of an RNA-DNA template-primer complexed with HIV-1 RT is unknown. The data presented here suggest that the configuration of an RNA-DNA helix complexed with HIV-1 RT differs from that of a DNA-DNA helix complexed with HIV-1 RT.
HIV-1 containing the M184V substitution is not only resistant to dideoxyinosine and dideoxycytidine, but is also resistant to the 1-␤-L-thiacytidine analogs 1-␤-L-FTC and 3TC (13)(14)(15). Faraj et al. (20) demonstrated that the K i values of M184V RT for 1-␤-L-ddCTP, 1-␤-L-FddCTP, and 3TCTP with poly(I)⅐(dC) 10 -15 as the template-primer are all significantly larger than the wt RT values, correlating with the significant increases (Ͼ2000fold) in the IC 50 values of M184V HIV-1 for 1-␤-L-FTC and 3TC. We used steady-state and pre-steady-state analyses to determine if RT containing the substitution at position 184 to valine had elevated K i values with respect to wt RT for 1-␤-L-cytidine analogs exclusively or 1-␤-L-nucleoside analogs in general. To begin to address these questions, a series of 1-␤-L-nucleoside analogs were studied: three pairs of cytidine analogs (which included thiacytidine analogs), two pairs of thymidine analogs, and one pair of guanosine analogs. The K i values of M184V RT for all 1-␤-L-nucleoside analogs determined were larger than those of wt RT. Interestingly, the effect of the valine substitution on the K i values for 1-␤-L-nucleoside analogs was not limited to RNA-directed DNA synthesis, as was observed with M184A RT for dNTPs and M184V RT for 1-␤-D-2Ј,3Јdideoxynucleotides. The data obtained with 1-␤-L-nucleoside analogs indicate that the M184V mutation allows the reverse transcriptase to become highly enantioselective and suggest that virus containing this mutation would be resistant to any 1-␤-L-nucleoside analog, regardless of substitutions in the sugar or base portions of the nucleoside. In contrast, the selectivity of M184A RT suggests that virus containing this mutation would not be enantioselective.
1-␤-L-Nucleoside analogs are alternate substrates for wt RT (17,20,31). To address this question with regard to M184V RT, an incorporation experiment was performed in which 1-␤-Dand 1-␤-L-ddCTP, 3TCTP, and FTCTP were incubated with wild-type and mutant enzymes bound to r44⅐[ 32 P]d23-mer for a fixed time. All three 1-␤-L-cytidine analogs were substrates for the mutant enzyme; however, it would appear from this experiment that 1-␤-L-ddCTP is not as good a substrate for M184V RT compared with 3TCTP and 1-␤-L-FTCTP, even though the K i values determined under steady-state conditions were similar. The decreased substrate efficiency of 1-␤-L-ddCTP relative to 3TCTP and 1-␤-L-FTCTP could be the result of an increased K m , decreased k cat , or a combination of both of these parameters. In contrast to our results, Faraj et al. (20) have reported that 1-␤-L-ddCTP is not a substrate for M184V RT. This difference is most likely due to the differences in concentration of 1-␤-L-ddCTP used in each study. In the study reported by Faraj et al., the concentration of 1-␤-L-cytidine analogs in both the wt  RT and M184V RT assays was 1 M; the K i value of M184V RT was 60-fold larger than that of wt RT (120 and 2.0 M, respectively). In the assay reported here, the concentration of the 1-␤-L-cytidine analogs in the mutant RT assays was 20-fold higher than in the wt RT assays. If k cat values were similar for wt RT and M184V RT, then under the assay conditions of Faraj et al., M184V RT would have 1.7% of the activity of wt RT for 1-␤-L-ddCTP turnover, which, in the 15-min time course of the assay, could account for the observed lack of 1-␤-L-ddCTP turnover with M184V RT.
Steady-state data for dCTP and 1-␤-L-FTCTP indicated that the two substrates had similar K m values for wt RT; with r44⅐d23-mer as the template-primer, the K m value for dCTP was 70 nM, and that for 1-␤-L-FTCTP was 130 nM. However, pre-steady-state kinetic analysis demonstrated that, even though the steady-state K m values were similar, the K d and k p values for the two substrates were very different. The K d value for dCTP (16 M) was ϳ10-fold higher than that for 1-␤-L-FTCTP (1.7 M); therefore, 1-␤-L-FTCTP is a tighter binding substrate than dCTP. The observed first-order rate constant (k p ) for dCTP was 9 s Ϫ1 , and that for 1-␤-L-FTCTP was 0.24 s Ϫ1 , yielding k p /K d values of 0.56 and 0.14 s Ϫ1 M Ϫ1 , respectively. The K m value for substrate during forced termination of processive synthesis is k off (K d /k p ); the K m values of wt RT for dCTP and 1-␤-L-FTCTP were similar because the respective k p /K d ratios were similar.
Steady-state data for M184A RT suggested that a mutation at this position affected the K d value for nucleotide substrate. This was confirmed using pre-steady-state kinetic analysis; the mutation to valine at Met-184 resulted in a 2-fold increase in K d for dCTP and a 20-fold increase in K d for 1-␤-L-FTCTP. Therefore, the increase in IC 50 values of HIV-1 M184V for 1-␤-L-nucleoside analogs correlates with a significant increase in the equilibrium binding constant specifically for the 1-␤-L-substrate. However, when comparing the K d values of M184V RT for dCTP and 1-␤-L-FTCTP, it was not evident as to why the K m value for 1-␤-L-FTCTP was higher than that for dCTP; the mutant enzyme bound 1-␤-L-FTCTP as well as it bound dCTP. It was only clear when the k p values for the two compounds were compared. Turnover of substrate by M184V RT into the growing strand of DNA was 50-fold slower for 1-␤-L-FTCTP than for dCTP, resulting in k p /K d ratios of M184V RT for dCTP of 0.23 s Ϫ1 M Ϫ1 and for 1-␤-L-FTCTP of 0.0056 s Ϫ1 M Ϫ1 . Thus, 1-␤-L-FTCTP was a less efficient substrate than dCTP of M184V RT because the k p /K d ratio was 40-fold lower.
The data presented in this study are further evidence that the YMDD region is important in catalysis for HIV-1 RT. Specifically, the amino acid at position 184 in the YMDD loop directly participates in nucleotide substrate binding.