Moulard M, Chaloin L, Canarelli S, Mabrouk K, Darbon H
C.I.M.L., Marseille, France, Laboratoire AFMB, IBSM, Marseille, France.
Biochemistry. 1998 Mar 31;37(13):4510-7. doi: 10.1021/bi972662f.
Proteolytic activation of retroviral envelope glycoprotein precursors occurs at the carboxyl side of a consensus motif consisting of the amino acid sequence (Arg/Lys)-Xaa-(Arg/Lys)-Arg. Synthetic peptides spanning the processing sites of HIV-1/2 and SIV glycoprotein precursors were examined for their ability to be cleaved by the subtilisin-like endoproteases kexin and furin. To determine the potential role of secondary structure on proteolytic activation, we examined the secondary structure of synthetic peptides by circular dichroism and NMR spectroscopy. The results indicate that (i) the peptides were correctly cleaved by kexin and furin and therefore could be used as specific substrates for the purification and characterization of the lymphocyte endoprotease(s) responsible for proteolytic processing of precursors; (ii) the regions surrounding the cleavage sites could be characterized by their flexibility in aqueous solutions. However, a loop has been shown to be a determinant for the specificity of the interaction between the enzyme and its substrate as determined by molecular modeling. Furthermore, we determine and propose a possible structure of the cleavage site which fits to the active site of the modeled furin.
逆转录病毒包膜糖蛋白前体的蛋白水解激活发生在由氨基酸序列（精氨酸/赖氨酸）-Xaa-（精氨酸/赖氨酸）-精氨酸组成的共有基序的羧基侧。检测了跨越HIV-1/2和SIV糖蛋白前体加工位点的合成肽被枯草杆菌蛋白酶样内切蛋白酶克新和弗林蛋白酶切割的能力。为了确定二级结构对蛋白水解激活的潜在作用，我们通过圆二色性和核磁共振光谱研究了合成肽的二级结构。结果表明：（i）这些肽被克新和弗林蛋白酶正确切割，因此可用作负责前体蛋白水解加工的淋巴细胞内切蛋白酶纯化和表征的特异性底物；（ii）切割位点周围的区域可通过其在水溶液中的灵活性来表征。然而，通过分子建模确定，一个环已被证明是酶与其底物之间相互作用特异性的决定因素。此外，我们确定并提出了一个适合建模的弗林蛋白酶活性位点的切割位点可能结构。
