Reardon J E, Spector T
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1989 May 5;264(13):7405-11.
Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.
阿昔洛韦三磷酸酯（ACVTP）是单纯疱疹病毒1型（HSV-1）DNA聚合酶的底物，并迅速掺入到一个合成模板引物中，该模板引物设计用于接受dGTP或ACVTP，随后是dCTP。ACVTP不会使HSV-1 DNA聚合酶失活，3'-末端带有阿昔洛韦单磷酸部分的模板引物也不是强效抑制剂。在将阿昔洛韦单磷酸掺入引物的3'-末端后，当模板编码的下一个脱氧核苷5'-三磷酸结合时，观察到对HSV-1 DNA聚合酶的强效抑制。dCTP（“下一个核苷酸”）从这个终产物复合物中解离的Ki为76 nM。相比之下，dCTP作为底物掺入到3'-引物末端含有dGMP而非阿昔洛韦单磷酸的模板引物中的Km为2.6 μM。下一个核苷酸有效结合的结构要求表明，一系列类似物的抑制效力顺序为：dCTP远大于阿拉伯糖基-CTP大于2'-3'-二脱氧-CTP远大于CTP、dCMP、dCMP + PPi。在存在下一个所需脱氧核苷酸（dCTP）的情况下，高浓度的dGTP与ACVTP竞争结合，从而阻碍终产物复合物的形成。这导致酶活性的一级损失，与基于机制的失活剂预期的情况无法区分。通过稳态动力学分析、分析凝胶过滤以及通过Sephadex G-25的快速凝胶过滤证明了终产物复合物的可逆性。研究表明，当使用聚（dC）-寡聚（dG）或活化的小牛胸腺DNA作为模板引物时，ACVTP和下一个所需的脱氧核苷5'-三磷酸也会发生强效、可逆的抑制作用。
