Pickering T J, Garforth S J, Thorpe S J, Sayers J R, Grasby J A
Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
Nucleic Acids Res. 1999 Feb 1;27(3):730-5. doi: 10.1093/nar/27.3.730.
Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.
噬菌体T5 5'→3'核酸外切酶是一类与序列相关的5'核酸酶家族的成员，这些核酸酶在DNA复制中起着至关重要的作用。5'核酸酶具有核酸外切酶活性和结构特异性内切核酸酶DNA切割活性，在噬菌体和哺乳动物等多种生物中都很保守。在此，我们报道了一种针对该酶的结构特异性单切割测定法的开发，该方法使用5'突出端发夹底物。DNA水解产物通过质谱进行表征。报道了该酶的稳态催化参数，得出结论：噬菌体T5 5'→3'核酸外切酶将特定磷酸二酯键的切割加速了至少10^15倍。催化测定已扩展到噬菌体T5 5'→3'核酸外切酶的三个突变体，即K83A、K196A和K215A。这三个赖氨酸残基中的任何一个突变为丙氨酸都会损害催化效率。所有三个赖氨酸都有助于底物的基态结合。此外，K83在该酶催化的化学反应中起重要作用。讨论了突变赖氨酸残基的可能作用。
