Crone T M, Goodtzova K, Pegg A E
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033-0850, USA.
Mutat Res. 1996 May 15;363(1):15-25. doi: 10.1016/0921-8777(95)00058-5.
Amino acid residues in the human O6-alkylguanine-DNA alkyltransferase (AGT) were mutated and seventeen of the mutant proteins expressed in the ada- ogt-E. coli strain GWR 109 which is very sensitive to killing by methylating agents because of the absence of endogenous alkyltransferases. Thirteen of the mutations tested (delta-10, delta 1-19, R128A, N137A, H146A, R147A, delta N157, Y158A, E172Q, delta 92-97, Y114E, C145A and E172stop) reduced activity to below detectable levels when crude cell extracts were tested for the ability to remove O6-[3H]methylguanine from 3H-methylated DNA. However, only 4 of these mutations (delta 92-97, Y114E, C145A and E172stop) led to a complete loss of activity when tested for the ability to protect the cells from killing by MNNG. This suggests that the other nine mutations do not lead to the complete inactivation of AGT but produce protein with a reduced activity or in reduced amounts. These results show that none of the residues altered in these mutations (delta 1-10, delta 1-19, R128A, N137A, delta N157, H146A, R147A, Y158A and E172Q) are absolutely essential for AGT activity in protection against killing by MNNG. The stability of the mutant AGT proteins was determined by measuring the half-life of the protein synthesis was blocked. These results indicated that five of mutants that lacked AGT activity when tested in the crude extracts (Y114E, R128A, C145A, delta N157 and Y158A) were stable in the cell showing that the alteration of these residues does greatly reduce AGT activity. The other eight mutants lacking activity in crude extracts (delta 1-10, delta 92-97, E172Q, E172stop, delta 1-19, N137A, H146A and R147A) produced a large decrease in the stability of the AGT protein. This may account for the inability to detect AGT activity in vitro despite the ability to protect from MNNG toxicity in vivo. It is of particular interest that mutation of residues His146, Arg147, Asn137 and Glu172 resulted in unstable AGT proteins active in vivo but not in vitro. The crystal structure of the related Ada-C alkyltransferase suggests the involvement of these residues with the Cys145 acceptor site in a hydrogen bond network that may stabilize the protein and aid in the reaction mechanism. The data presented here support the existence of such an interaction existing in the human AGT and stress its importance in maintaining the configuration of the protein.
人O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）中的氨基酸残基发生了突变，其中17种突变蛋白在ada-ogt-E. coli菌株GWR 109中表达，该菌株由于缺乏内源性烷基转移酶，对甲基化剂杀伤作用非常敏感。测试的13种突变（δ-10、δ1-19、R128A、N137A、H146A、R147A、δN157、Y158A、E172Q、δ92-97、Y114E、C145A和E172stop）在粗细胞提取物测试从3H-甲基化DNA中去除O6-[3H]甲基鸟嘌呤的能力时，活性降低到检测不到的水平以下。然而，当测试这些突变体保护细胞免受MNNG杀伤的能力时，其中只有4种突变（δ92-97、Y114E、C145A和E172stop）导致活性完全丧失。这表明其他9种突变不会导致AGT完全失活，而是产生活性降低或数量减少的蛋白质。这些结果表明，这些突变（δ1-10、δ1-19、R128A、N137A、δN157、H146A、R147A、Y158A和E172Q）中改变的残基对于AGT在保护细胞免受MNNG杀伤中的活性并非绝对必需。通过测量蛋白质合成被阻断后的半衰期来确定突变AGT蛋白的稳定性。这些结果表明，在粗提取物测试中缺乏AGT活性的5种突变体（Y114E、R128A、C145A、δN157和Y158A）在细胞中是稳定的，表明这些残基的改变并没有大大降低AGT活性。在粗提取物中缺乏活性的其他8种突变体（δ1-10、δ92-97、E172Q、E172stop、δ1-19、N137A、H146A和R147A）导致AGT蛋白的稳定性大幅下降。这可能解释了尽管在体内能够保护细胞免受MNNG毒性，但在体外却无法检测到AGT活性的原因。特别有趣的是，His146、Arg147、Asn137和Glu172残基的突变导致AGT蛋白在体内有活性但在体外无活性且不稳定。相关Ada-C烷基转移酶的晶体结构表明，这些残基与Cys145受体位点参与了一个氢键网络，该网络可能稳定蛋白质并有助于反应机制。这里呈现的数据支持了人类AGT中存在这种相互作用，并强调了其在维持蛋白质构象中的重要性。
