Capson T L, Peliska J A, Kaboord B F, Frey M W, Lively C, Dahlberg M, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1992 Nov 17;31(45):10984-94. doi: 10.1021/bi00160a007.
The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.
噬菌体T4的DNA聚合酶是DNA合成所需的多酶复合物的一部分。作为了解单个蛋白质对复合物动态特性（如周转、持续合成能力和复制保真度）贡献的第一步，已通过预稳态动力学方法确定了基因43蛋白的聚合酶和核酸外切酶活性的最小动力学方案，并通过计算机模拟进行拟合。使用DNA引物/模板（13/20聚体）作为底物；含有更多单链DNA的双链体导致聚合酶的无效结合。反应序列的特征是13/20聚体和dATP按顺序添加到T4酶上（解离常数分别为70 nM和20 μM），随后T4.13/20聚体.dATP复合物快速转化（400 s-1）为T4.14/20聚体.PPi产物种类。PPi释放后的一个慢步骤（2 s-1）限制了单次周转，尽管在以>400 s-1的速率发生的多次掺入（13/20聚体→17/20聚体）中该步骤被绕过。相对于模板链，正确与错误核苷酸之间的竞争表明，错误核苷酸的解离常数处于毫摩尔值，因此提供了证据表明，T4聚合酶与T7聚合酶一样，但与Klenow片段聚合酶不同，在核苷酸结合步骤中对错误掺入的辨别因子>103。T4酶的核酸外切酶活性需要一个激活步骤，即T4.DNA→T4.(DNA)*，其速率常数反映引物的3'末端是匹配还是错配；对于匹配的13/20聚体，常数为1 s-1，对于错配的13T/20聚体，为5 s-1。交叉实验提供的证据表明，该步骤可能代表双链体末端的解链，随后是快速的核酸外切酶切割（100 s-1）。在存在正确的dNTP时，引物延伸是限速步骤，而不是涉及双链体在分开的核酸外切酶和聚合酶位点之间移动的步骤。由于13/20聚体或13T/20聚体从酶上解离的速率常数为6或8 s-1，并与激活速率常数竞争，因此酶单独单次通过进行的核酸外切酶编辑效率有点低（5 s-1/(8 s-1+5 s-1)），约为40%。因此，辅助蛋白的主要作用可能是减缓酶-底物解离的速率，从而提高整体保真度和持续合成能力。
