St Hilaire P M, Willert M, Juliano M A, Juliano L, Meldal M
Department of Chemistry, Carlsberg Laboratory, Valby-Copenhagen, Denmark.
J Comb Chem. 1999 Nov-Dec;1(6):509-23. doi: 10.1021/cc990031u.
To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.
为了绘制半胱氨酸蛋白酶的底物特异性图谱，合成了两个组合肽库，并使用原型蛋白酶木瓜蛋白酶进行筛选。使用聚乙二醇酸树脂作为库合成的固相支持物，便于应用树脂上荧光猝灭测定法。对库2的筛选结果表明，S3亚位点偏好脯氨酸或缬氨酸，S2偏好疏水残基；最常见的残基不是苯丙氨酸而是缬氨酸。S1亚位点对小的非极性残基丙氨酸或甘氨酸以及较大的谷氨酰胺和带电荷的残基精氨酸表现出双重特异性。小残基在S1'-S4'亚位点占主导。重新合成了来自库及其变体的活性肽，并在溶液相测定中评估了它们被木瓜蛋白酶水解的动力学。一般来说，固相上底物切割的程度与溶液相测定中获得的kcat/KM值之间有很好的相关性。获得了几种木瓜蛋白酶的良好底物，最佳底物是Y(NO2)PMPPLCTSMK(Abz)（kcat/KM = 2109 (mM s)-1）、Y(NO2)PYAVQSPQK(Abz)（kcat/KM = 1524 (mM s)-1）和Y(NO2)PVLRQQRSK(Abz)（kcat/KM = 1450 (mM s)-1）。通过使用分子动力学（MD）从结构角度解释了这些结果。这些MD计算表明底物在狭窄酶裂隙中的结合有两种不同模式。
