Jang G F, Gelb M H
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Biochemistry. 1998 Mar 31;37(13):4473-81. doi: 10.1021/bi972289b.
We have previously identified proteolytic activity in rat liver microsomes that cleaves an intact tripeptide, VIS, from S-farnesylated-CVIS tetrapeptide. This enzymatic activity, termed prenyl protein-specific endoprotease (PPEP) activity, has been solubilized in CHAPS and purified 5-fold. To probe the peptide recognition features of PPEP activity, 64 tripeptides [N-acetyl-C(S-farnesyl)a1a2] were prepared and tested as competitive inhibitors of PPEP activity-catalyzed hydrolysis of N-acetyl-C(S-farnesyl)VI[3H]S. It was found that PPEP activity prefers large hydrophobic residues in the a1 and a2 positions. A subset of N-acetyl-C(S-farnesyl)a1a2 peptides were prepared in radiolabeled form, and it was found that PPEP activity preferences for these substrates correlated well in most cases with the inhibition data. The exception is that R in the a1 position does not prevent binding of peptide to PPEP activity, but such peptides are poor substrates. The anionic residue D in the a2 position is not tolerated by PPEP activity. Five farnesylated radiolabeled tetrapeptides, Ac-C(F)FM[3H]L, Ac-C(F)LI[3H]L, Ac-C(F)LL[3H]L, Ac-C(F)LM[3H]L, and Ac-C(F)VI[3H]L were prepared, and PPEP activity kinetic studies revealed that they are good substrates and show comparable KM values (2.2-13.5 microM). Ac-C(F)RL[3H]S is a poor substrate. The reported peptide binding preferences of PPEP activity should be useful in designing compounds that block the C-terminal proteolysis of prenylated proteins. Nonprenylated peptides do not bind to PPEP activity, and replacement of the farnesyl group with ann-pentadecyl group modestly reduces binding. Peptide-membrane partitioning studies were used together with theoretical arguments to fully understand the substrate specificity of PPEP activity toward these compounds.
我们之前在大鼠肝脏微粒体中鉴定出一种蛋白水解活性，它能从法尼基化的CVIS四肽上切割下完整的三肽VIS。这种酶活性被称为异戊二烯基蛋白特异性内切蛋白酶（PPEP）活性，已在CHAPS中溶解并纯化了5倍。为了探究PPEP活性的肽识别特征，制备了64种三肽[N-乙酰基-C（法尼基）a1a2]，并作为PPEP活性催化的N-乙酰基-C（法尼基）VI[3H]S水解的竞争性抑制剂进行测试。发现PPEP活性在a1和a2位置更喜欢大的疏水残基。制备了一组放射性标记形式的N-乙酰基-C（法尼基）a1a2肽，发现在大多数情况下，PPEP活性对这些底物的偏好与抑制数据相关性良好。例外情况是，a1位置的R并不妨碍肽与PPEP活性的结合，但此类肽是较差的底物。PPEP活性不能容忍a2位置的阴离子残基D。制备了五种法尼基化的放射性标记四肽，Ac-C（F）FM[3H]L、Ac-C（F）LI[3H]L、Ac-C（F）LL[3H]L、Ac-C（F）LM[3H]L和Ac-C（F）VI[3H]L，PPEP活性动力学研究表明它们是良好的底物，并且显示出相当的KM值（2.2 - 13.5 microM）。Ac-C（F）RL[3H]S是较差的底物。所报道的PPEP活性的肽结合偏好对于设计阻断异戊二烯基化蛋白C末端蛋白水解的化合物应该是有用的。非异戊二烯基化的肽不与PPEP活性结合，用正十五烷基取代法尼基基团会适度降低结合。肽-膜分配研究与理论论证一起用于全面理解PPEP活性对这些化合物的底物特异性。
