Kuchta R D, Mizrahi V, Benkovic P A, Johnson K A, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1987 Dec 15;26(25):8410-7. doi: 10.1021/bi00399a057.
The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.
利用特定序列的短DNA寡聚物，已经确定了大肠杆菌DNA聚合酶I（KF）的Klenow片段催化DNA聚合反应的最小动力学机制。该机制的一个关键特征是一个最小的两步序列，它能使三元复合物KF.DNAn.dNTP和KF.DNAn + 1.PPi相互转化。反应速率不受实际聚合反应的限制，而是受一个单独步骤的限制，这一步骤可能对确保保真度很重要[米兹拉希，V.，亨利，R. N.，马利尔，J. F.，约翰逊，K. A.，& 本科维奇，S. J.（1985年）《生物化学》24卷，4010 - 4018页]。单周转焦磷酸解实验（聚合反应的微观逆反应）中双相动力学的观察结果为这一序列提供了证据。数据分析进而给出了内部平衡常数的估计值。通过分配（同位素捕获）实验测量了DNA、dNTP和PPi从各种二元和三元复合物中的解离情况。DNA从KF上解离的速率常数与序列有关，并且在非连续DNA合成过程中是限速的。单周转（两个方向）和同位素捕获实验的结合提供了足够的信息，以便对特定DNA序列的动力学机制进行定量评估。
