Landro J A, Raybuck S A, Luong Y P, O'Malley E T, Harbeson S L, Morgenstern K A, Rao G, Livingston D J
Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts 02139-4242, USA.
Biochemistry. 1997 Aug 5;36(31):9340-8. doi: 10.1021/bi963054n.
Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.
丙型肝炎病毒（HCV）感染是一个严重的医学问题，对于慢性肝炎的进展尚无广泛有效的治疗方法。位于非结构蛋白3（NS3）N端三分之一区域的病毒丝氨酸蛋白酶的催化活性，对于多蛋白在四个位点特异性连接处的加工是必需的。NS3-NS4A共复合物的三维晶体结构[Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343 - 355]描绘了54个氨基酸残基的NS4A蛋白内的一个小疏水区域，该区域插入蛋白酶核心并与其广泛接触。当前的研究利用一个小的合成NS4A肽（病毒多蛋白序列的1678 - 1691位残基）和重组表达的NS3蛋白酶结构域，探讨了NS4A激活NS3蛋白酶催化活性的机制。当以代表多蛋白不同位点特异性连接处的小肽底物进行测定时，添加NS4A显著增加了NS3的催化参数kcat和kcat/Km。研究了5A/5B肽底物中P1位天然和非天然氨基酸取代的催化效应。NS3 - NS4A对真实底物中常见的半胱氨酸残基表现出明显的催化偏好。NS4A的存在不影响NS3水解反应的pH依赖性。该结果表明NS4A不会改变NS3蛋白酶活性位点残基的pKa值。进行了稳态动力学分析，结果表明在催化循环中，NS4A和肽底物以有序方式结合，NS4A先结合。基于5A/5B切割位点鉴定出两类不同动力学性质的肽基抑制剂。一类不依赖NS4A的抑制剂没有引发残基。第二类抑制剂依赖NS4A，并且含有一个天然或非天然的环状氨基酸取代了常见的P1'位丝氨酸残基。在NS4A存在的情况下，这些抑制剂对NS3蛋白酶的亲和力增加高达80倍。对这类抑制剂的引发残基和P残基进行连续截短，证明了P4'和P1'残基对于有效抑制至关重要。利用与NS3 - NS4A结构活性位点结合的5A/5B十肽底物模型，解释了这种NS4A效应的选择性。
