Bulychev N V, Varaprasad C V, Dormán G, Miller J H, Eisenberg M, Grollman A P, Johnson F
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Biochemistry. 1996 Oct 8;35(40):13147-56. doi: 10.1021/bi960694h.
The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme. In contrast to a recent report [Lu, A.-L., et al. (1995) J. Biol. Chem. 270, 23582], dA:8-oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (kcat/Km) for dA:8-oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min-1 nM-1), respectively. kcat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 x 10(-6) (min-1 nM-1), whereas replacement of dA by 2'-O-methyladenosine virtually abolished enzymatic activity. Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity constant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate. Duplexes containing a carbocyclic analog of dA were not cleaved. A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.
大肠杆菌的MutY蛋白可从DNA中去除错配的脱氧腺嘌呤残基。在本研究中，含有修饰碱基的双链寡脱氧核苷酸被用作该酶的模型底物。与最近的一份报告[Lu, A.-L., 等人(1995) J. Biol. Chem. 270, 23582]相反，dA:8-氧代-dG似乎是MutY的首选天然底物，dA:8-氧代-dG和dA:dG的特异性常数（kcat/Km）分别为39600×10⁻⁶和383×10⁻⁶（分钟⁻¹纳摩尔⁻¹），这证明了这一点。含有dA:dG的双链体的kcat在较低pH值时最高；含有dA:8-氧代-dG的双链体在5.5 - 8.0的pH范围内切割速率不受影响。dG中8-氧代功能的存在显著提高了从所有测试底物中去除dA的速率。用rA取代dA将dA:8-氧代-dG的特异性常数降低到294×10⁻⁶（分钟⁻¹纳摩尔⁻¹），而用2'-O-甲基腺苷取代dA实际上消除了酶活性。dG部分的修饰通常比dA的修饰更能耐受；然而，在dG的6位引入甲基醚产生了一种不可切割的底物，用2'-O-甲基鸟苷取代dG产生了一种特异性常数低的底物。含有dA:dC和dA:四氢呋喃的双链体的切割速率比参考底物低三个数量级。含有dA的碳环类似物的双链体未被切割。提出了一个模型来解释MutY对DNA底物的识别以及该酶的催化特性。
