噬菌体RB69 DNA聚合酶以不正确的核苷酸为底物的聚合反应的稳态动力学研究

测定了ＲＢ６９ ＤＮＡ 聚合酶以不正确的核苷酸（ｒＮＴＰ、ｄｄＮＴＰ以及碱基不配对的ｄＮＴＰ）为底物进行聚合反应的稳态动力学常数，并与Ｋｌｅｎｏｗ 酶进行了比较．结果表明，ＲＢ６９ ＤＮＡ 聚合酶在以不正确的核苷酸为底物进行聚合反应时，其Ｋｍ 值与正确底物参入时相比有大幅度提高，而ｋｃａｔ保持不变或下降幅度较小．而Ｋｌｅｎｏｗ 酶在利用不正确的核苷酸为底物时，与正确底物参入时相比，其ｋｃａｔ大幅度下降，而Ｋｍ （或ＫＤ）基本保持不变或上升较小幅度．两种酶不同的动力学特点反映出它们不同的底物选择机制．

Characterization of Kinetic Parameters for rNTP/ddNTP Incorporation and dNTP Misincorporation by Bacteriophage RB69 DNA Polymerase

The steady state kinetic parameters for rNTP/ddNTP incorporation and dNTP misincorporation by bacteriophage RB69 DNA polymerase have been determined, and compared with the steady state or pre steady state data from Klenow fragment, RB69 DNA polymerase selected correct incoming dNTP against incorrect nucleotides by largely increasing the K m values for incorrect nucleotides but keeping k cat values unchanged or slightly decreased, while Klenow fragment by largely decreasing k cat values but keeping K m values unchanged or slightly increased. The kinetic difference between the two polymerases reflected their different substrate selection mechanism.