Gutierrez J C, Götze O, Caporale L H
Biochim Biophys Acta. 1983 May 18;744(3):276-80. doi: 10.1016/0167-4838(83)90200-5.
By lengthening the sequence of a previously available tripeptide to a pentapeptide, we were able to increase the specificity of the substrate for the complement enzyme CVFBb (EC 3.4.21.47) over Factor Xa (EC 3.4.21.6). This increase in specificity was achieved by both an increase in the kcat/Km for CVFBb for the longer substrate compared to the original substrate, and a decrease in the kcat/Km for Factor Xa. The new substrate, Boc-Nle-Gln-Leu-Gly-Arg-amino methyl coumarin (AMC) was synthesized by coupling Boc-Nle-Gln-Leu-Gly to Arg-AMC in solution. p-Toluenesulfonic acid was added to the coupling mixture to improve the solubility of the arginine derivative and avoid the need for covalent protection.
通过将先前可得的三肽序列延长为五肽，我们能够提高底物对补体酶CVFBb（EC 3.4.21.47）相对于因子Xa（EC 3.4.21.6）的特异性。这种特异性的提高是通过与原始底物相比，较长底物对CVFBb的kcat/Km增加以及因子Xa的kcat/Km降低来实现的。新底物Boc-Nle-Gln-Leu-Gly-Arg-氨基甲基香豆素（AMC）是通过在溶液中将Boc-Nle-Gln-Leu-Gly与Arg-AMC偶联合成的。向偶联混合物中加入对甲苯磺酸以提高精氨酸衍生物的溶解度并避免共价保护的需要。
