Lu A L, Yuen D S, Cillo J
Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Maryland 21201, USA.
J Biol Chem. 1996 Sep 27;271(39):24138-43. doi: 10.1074/jbc.271.39.24138.
Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.
大肠杆菌MutY蛋白通过N-糖基化酶以及脱嘌呤/脱嘧啶内切核酸酶或裂解酶活性，在A链上切割含A/G或a/7,8-二氢-8-氧代鸟嘌呤（A/GO）的DNA。在本文中，我们表明在硼氢化钠存在的情况下，MutY能够被困在一种稳定的共价酶-DNA中间体中，这一新发现支持将MutY归为具有伴随脱嘌呤/脱嘧啶裂解酶活性的那一类DNA糖基化酶。为了潜在地帮助确定MutY的底物识别位点，构建了突变蛋白。具有Gly116→Ala（G116A）或Asp（G116D）突变的MutY蛋白对含A/G和含A/GO的DNA底物的结合亲和力降低。然而，催化参数受到的影响有所不同。虽然MutY切割含A/G和含A/GO的DNA的特异性常数（kcat/Km）分别为10和3.3 min-1μM-1，但MutY（G116D）切割这些DNA的效率分别降低了2倍、300倍和9倍。MutY（G116A）对含A/G和含A/GO的DNA的催化活性与野生型MutY大致相同。在硼氢化钠存在的情况下，MutY（G116A）和MutY（G116D）都能与含A/GO的DNA被困在共价中间体中，但效率低于野生型酶。MutY（G116A）也能与含A/G的DNA形成共价中间体，但MutY（G116D）不能。由于MutY的Gly116位于几种DNA糖基化酶中高度保守的区域，这个保守区域可能靠近底物结合和/或催化位点。
