Yokoyama K, Zimmerman K, Scholten J, Gelb M H
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195-1700, USA.
J Biol Chem. 1997 Feb 14;272(7):3944-52. doi: 10.1074/jbc.272.7.3944.
Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.
蛋白质香叶基香叶基转移酶-I（PGGT-I）和蛋白质法尼基转移酶（PFT）分别将香叶基香叶基和法尼基基团连接到真核细胞蛋白质的C末端。在体外，PGGT-I和PFT可以将香叶基香叶基焦磷酸（GGPP）和法尼基焦磷酸（FPP）中的香叶基香叶基和法尼基基团转移到它们的蛋白质或肽类异戊二烯基受体底物上。本研究表明，PGGT-I与GGPP的结合比与FPP的结合紧密330倍，而PFT与FPP的结合比与GGPP的结合紧密15倍。因此，在体内，当GGPP和FPP竞争与异戊二烯基转移酶的结合时，PGGT-I和PFT可能分别主要与GGPP和FPP结合。先前的研究表明，K-Ras4B和Ras相关的GTP酶TC21在体外是PGGT-I和PFT的底物。结果表明，TC21可以分别与异三聚体G蛋白γ亚基的C末端肽和核纤层蛋白B的C末端肽竞争，从而分别阻止PGGT-I的香叶基香叶基化和PFT的法尼基化。在这两种情况下K-Ras4B都能竞争，但在存在核纤层蛋白B肽竞争者的情况下，它几乎完全被PFT法尼基化。快速和单周转动力学研究表明，反应循环中PGGT-I催化的香叶基香叶基转移步骤的速率常数比稳态周转数大14倍，这表明总反应速率受异戊二烯基转移后续步骤（如产物从酶中释放）的限制。PGGT-I催化的法尼基化比香叶基香叶基化慢37倍，并且受法尼基转移步骤的限制。这些结果与早期研究共同为PGGT-I和PFT的底物特异性提供了范例，并为设计作为抗癌药物的异戊二烯基转移酶抑制剂提供了关键信息。
