Jaju M, Beard W A, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068, USA.
J Biol Chem. 1995 Apr 28;270(17):9740-7. doi: 10.1074/jbc.270.17.9740.
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T.P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T.P binary complex (RTT.P). 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)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Km should be equivalent to Ki, and since Km for AZTTP should be influenced by the dissociation rate constant for RTT.P, we examined the effect of altering RTT.P 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. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower steady-state phase representing kcat (0.42 min-1) which was similar to the rate constant for RTT.P dissociation. Additionally, Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT.P dissociation rate constant and Ki,AZTTP was not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT.P exist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T.P where the binary RTT.P complex undergoes an isomerization before binding of the deoxynucleotide substrate.
1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）通过有序的顺序机制催化DNA合成。模板引物（T.P）与游离酶结合后，要掺入的脱氧核苷三磷酸与RT和T.P二元复合物（RTT.P）结合。结合的核苷酸掺入后，催化循环受限于构象变化（用于持续合成）或酶从延伸的T.P释放（用于单核苷酸掺入）。为了探索通过这些交替的限速步骤进行的循环，我们测定了HXB2R HIV-1 RT在同聚物T.P系统聚（rA）-寡聚（dT）16上利用链终止核苷酸底物3'-叠氮基-3'-脱氧胸苷三磷酸（AZTTP）和双脱氧胸苷三磷酸进行单核苷酸掺入的动力学参数。还研究了这些链终止底物对持续的脱氧胸苷单磷酸掺入的抑制作用。由于AZTTP是一种底物，其Km应等同于Ki，并且由于AZTTP的Km应受RTT.P解离速率常数的影响，我们研究了改变RTT.P解离对AZTTP动力学参数的影响。通过使用不同的T.P底物、RT的病毒来源以及在一个干扰T.P结合的残基处发生改变的突变RT来调节解离速率常数。正如早期工作所预期的那样，AZTMP在聚（rA）-寡聚（dT）16上的掺入时间进程是双相的，先是一个爆发期，随后是一个较慢的稳态期，代表kcat（0.42分钟-1），这与RTT.P解离的速率常数相似。此外，AZTTP的Km（110 nM）低于其平衡解离常数（1200 nM）。AZTTP对持续的dTMP掺入和单核苷酸掺入的抑制作用（Ki,AZTTP）相似。然而，未观察到RTT.P解离速率常数与Ki,AZTTP之间的简单相关性。这些结果表明，单核苷酸掺入的简单有序模型是不充分的，并且存在不同形式的RTT.P，它们可以限制催化作用。在T.P的两步结合反应的背景下讨论了这些结果，其中二元RTT.P复合物在脱氧核苷酸底物结合之前经历异构化。
