Bhattacharyya D, Hazra T K, Behnke W D, Chong P L, Kurosky A, Lee J C, Mitra S
University of Tennessee Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory, Tennessee 37831, USA.
Biochemistry. 1998 Feb 10;37(6):1722-30. doi: 10.1021/bi971852n.
The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains. The two domains are of nearly equal size and are connected by a hinge region. The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue. This protein functions in DNA repair by direct dealkylation of mutagenic O6-alkylguanine. The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 degrees C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity. However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation. Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein. This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure. The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine. These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.
多功能的39 kDa大肠杆菌Ada蛋白（O6-甲基鸟嘌呤-DNA甲基转移酶）（EC 2.1.1.63）是ada基因的产物，它是一种单体球状多肽，在两个结构域中具有两种不同的烷基受体活性。这两个结构域大小几乎相等，通过一个铰链区相连。Ada蛋白按化学计量从DNA中的O6-烷基鸟嘌呤的Cys-321残基以及从烷基磷酸三酯的Cys-69残基接受烷基。该蛋白通过对诱变的O6-烷基鸟嘌呤进行直接脱烷基作用参与DNA修复。在Cys-69处甲基化的蛋白成为ada操纵子中包括其自身基因的转录激活因子。两个结构域中的每一个都作为一个独立的烷基受体发挥作用。纯化的均一蛋白在37℃不稳定，在不到30分钟内自发失去约30%的二级结构，同时完全丧失活性。然而，沉降平衡研究表明，失活的蛋白仍保持单体形式，没有聚集。此外，电喷雾质谱分析表明失活的蛋白没有氧化现象。DNA可抑制Ada蛋白这种温度依赖性的失活。在尿素或胍浓度增加的情况下，该蛋白逐渐失去超过80%的结构。两种烷基受体活性对去折叠的敏感性似乎不同，磷酸三酯甲基转移酶活性对7 M尿素有抗性。通过去除变性剂，尿素或胍诱导的部分或完全去折叠在几秒钟内可完全逆转。热凝聚的蛋白也可以通过用8 M尿素或6 M胍处理循环恢复到完全活性。这些结果表明，新生的或未折叠的Ada多肽折叠成一种亚稳态形式，这种形式是有活性的，而热力学稳定结构是部分去折叠的且无活性。
