Wilson J E, Aulabaugh A, Caligan B, McPherson S, Wakefield J K, Jablonski S, Morrow C D, Reardon J E, Furman P A
Division of Biochemistry, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709, USA. 34294-0007, USA.
J Biol Chem. 1996 Jun 7;271(23):13656-62. doi: 10.1074/jbc.271.23.13656.
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.
通过将重组人免疫缺陷病毒1型逆转录酶（RT）中的甲硫氨酸184替换为丙氨酸（M184A）或缬氨酸（M184V）进行突变，并测定稳态和预稳态动力学常数。对于RNA指导的合成，M184A RT对dNTP的Km值大于野生型RT；对于持续合成或分布合成，M184A RT的kcat值相似。与M184A RT相反，M184V RT对dNTP底物的Km和kcat值与野生型RT相似。对于RNA和DNA指导的合成，M184V RT对1-β-L-核苷类似物的Ki值相对于野生型RT增加了30至500倍。根据单核苷酸掺入的预稳态动力学估算野生型RT和M184V RT对dCTP和顺式-5-氟-1-[2-(羟甲基)-1,3-氧硫杂环戊烷-5-基]胞嘧啶5'-三磷酸（1-β-L-FTCTP）的Kd和kp值。M184V RT对1-β-L-FTCTP的Kd值比野生型RT大19倍；两种酶的kp值相似。这些结果支持这样的假设，即野生型RT高度保守的YMDD区域中的甲硫氨酸184参与核苷（类似物）5'-三磷酸的结合。
