Gorecki M, Panet A
Biochemistry. 1978 Jun 13;17(12):2438-42. doi: 10.1021/bi00605a030.
The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.
禽成髓细胞瘤病毒逆转录酶的活性位点已被多种化学试剂选择性修饰。DNA聚合酶活性对疏水性巯基试剂如5,5'-二硫代双（2-硝基苯甲酸）和对羟基汞苯甲酸非常敏感，但对具有亲水性的巯基试剂具有抗性。另一方面，RNase H活性对疏水性和亲水性巯基试剂均具有抗性，这表明不存在RNase H活性所必需的半胱氨酰残基。N-乙基马来酰亚胺（NEM）是一种对氨基和巯基具有特异性的试剂，它会使DNA聚合酶和RNase H失活，其中RNase H活性的稳定性是DNA聚合酶活性的四倍。多核苷酸而非三磷酸核苷酸可保护逆转录酶的两种酶活性免受NEM的影响。由于用5,5'-二硫代双（2-硝基苯甲酸）对该酶进行预处理并不能阻止N-乙基马来酰亚胺与DNA聚合酶活性所需的残基发生反应，因此可能有两个不同的反应基团参与了这种酶活性。N-乙基马来酰亚胺抑制逆转录酶的pH曲线也表明，对于DNA聚合酶活性而言，有两个反应基团至关重要，其表观pKa分别为5.5和6.5。而对于RNase H活性，仅发现一个pKa为7.5的反应基团与之相关。
