Dahl Göran, Arenas Omar Gutiérrez, Danielson U Helena
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
Biochemistry. 2009 Dec 8;48(48):11592-602. doi: 10.1021/bi9016928.
The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.
丙型肝炎病毒（HCV）的非结构蛋白3（NS3）是一种双功能酶，其蛋白酶和螺旋酶功能分别位于单肽链的两个结构域中。由于这两种酶的人工单结构域形式都具有催化活性，因此几乎没有实验证据表明这种意外排列具有功能作用。我们观察到，低浓度的某些蛋白酶抑制剂可使来自HCV 1a基因型的全长NS3蛋白酶的活性提高多达100%，这取决于预孵育时间和所用抑制剂。增加离子强度、降低甘油浓度或降低pH值可使激活作用减弱，但不会消除，且在所有情况下，都会以活性显著丧失为代价。对于与NS4A辅因子片段融合的1b基因型NS3的人工蛋白酶结构域，未观察到激活现象。这种截短且共价修饰的酶形式活性低得多，并且与没有融合辅因子的全长NS3蛋白酶相比，表现出根本不同的催化特性。发现激活作用最合理的解释涉及两种酶构象之间的缓慢转变，这两种构象在催化能力和对抑制剂的亲和力方面有所不同。基于该假设推导的方程比简单竞争性抑制方程更能拟合实验数据。该机制可能涉及抑制剂诱导螺旋酶结构域稳定在一种增强蛋白酶活性的构象中，或者涉及蛋白酶中催化三联体的排列改善。所提出的记忆机制和推导方程对于这两种解释都是可行的，并且可以作为未来研究由抑制剂或其他配体激活的酶的基本框架。
