Gangurde Rajiv, Modak Mukund J
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
Biochemistry. 2002 Dec 10;41(49):14552-9. doi: 10.1021/bi0264216.
We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP. The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex. Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process. This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation. These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).
我们研究了四个活性位点羧酸盐在大肠杆菌DNA聚合酶I（克列诺片段）预聚合酶三元复合物形成过程中的作用，该复合物包含模板引物和脱氧核苷三磷酸（dNTP）。对天冬氨酸（Asp）705、谷氨酸（Glu）710、天冬氨酸882和谷氨酸883进行保守和非保守取代的九种突变酶的分析清楚地表明，两个具有催化活性的必需天冬氨酸，即天冬氨酸705和天冬氨酸882，是形成稳定三元复合物所必需的。在这两个谷氨酸中，只有谷氨酸710是三元复合物形成所必需的，而谷氨酸883不参与此过程。这项研究还揭示了克列诺片段在酶-模板-引物二元复合物和酶-模板-引物-dNTP三元复合物形成方面的两个有趣特性。这些特性是：（a）酶-模板-引物-dNTP三元复合物对添加高盐或模板引物挑战具有显著抗性；（b）克列诺片段在非催化性二价阳离子如Ca(2+)、Co(2+)、Ni(2+)和Zn(2+)存在的情况下形成三元复合物的能力。
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