Lillehaug J R, Kleppe K
Biochemistry. 1975 Mar 25;14(6):1221-5. doi: 10.1021/bi00677a020.
The kinetics of T4 polynucleotide kinase has been investigated at pH 8.0 and 37 degrees. Double reciprocal plots of initial rates vs. substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to the ordered sequential mechanism shown in eq 2 in the text for phosphorylation of a DNA molecule. Based on this mechanism the rate equation for the overall reaction was deduced and the various kinetic constants estimated. Hill plots indicated little or no interaction between active sites in the enzyme. The apparent Michaelis constants and V-max were determined at a fixed ATP concentration, 66 muM, for a number of different substrates varying in chain length, base composition, and nature of the sugar, and a wide variation was found. For the nucleoside 3'-monophosphates tested both the apparent Michaelis constant and V-max values were from approximately 2 to 5 times larger than for the corresponding oligonucleotide. The following orders were obtained with regard to apparent Michaelis constants and V-max for the nucleoside 3'-monophosphates investigated: Michaelis constant, rGP greater than rUp greater than rCp greater than rAp greater than dTp; V-max, rGp greater than rCp greater than rAp greater than dTp greater than rUp. Somewhat similar results were also obtained with the deoxyoligonucleotides tested.
已在pH 8.0和37摄氏度条件下研究了T4多核苷酸激酶的动力学。初始速率对底物浓度的双倒数作图以及产物抑制研究表明，该酶按照文中式2所示的有序序列机制进行反应，以实现DNA分子的磷酸化。基于此机制推导了总反应的速率方程，并估算了各种动力学常数。希尔作图表明该酶活性位点之间几乎没有相互作用。在固定ATP浓度66μM下，针对多种链长、碱基组成和糖性质不同的底物测定了表观米氏常数和Vmax，发现存在很大差异。对于所测试的核苷3'-单磷酸，表观米氏常数和Vmax值均比相应的寡核苷酸大约大2至5倍。在所研究的核苷3'-单磷酸的表观米氏常数和Vmax方面获得了以下顺序：米氏常数，rGP＞rUp＞rCp＞rAp＞dTp；Vmax，rGp＞rCp＞rAp＞dTp＞rUp。对所测试的脱氧寡核苷酸也获得了 somewhat similar results。（此处原文“somewhat similar results”可能有误，按字面直译为“有点相似的结果”，可根据实际情况修正）
