Furfine E S, Leban J J, Landavazo A, Moomaw J F, Casey P J
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 1995 May 23;34(20):6857-62. doi: 10.1021/bi00020a032.
Protein farnesyltransferase (FTase) catalyzes the prenylation of Ras and several other key proteins involved in cell regulation. The mechanism of the FTase reaction was elucidated by pre-steady-state and steady-state kinetic analysis. FTase catalyzed the farnesylation of biotinylated peptide substrate (BiopepSH) by farnesyl pyrophosphate (FPP) to an S-farnesylated peptide (BiopepS-C15). The steady-state kinetic mechanism was ordered. FTase bound FPP in a two-step process with an effective dissociation rate constant of 0.013 s-1 and an overall Kd of 2.8 nM. BiopepSH reacted with FTase.FPP irreversibly, with a second-order rate constant of 2.2 x 10(5) M-1 s-1, to form FTase.BiopepS-C15. Because most of the FPP in FTase.FPP was trapped as FTase.BiopepS-C15 at high concentrations of BiopepSH, FPP dissociated slowly from the ternary complex relative to catalysis, so that the commitment to catalysis was high. The maximal rate constant for formation of FTase.BiopepS-C15 (enzyme-bound product) is much larger than kcat (0.06 s-1), indicating that product release is the rate-determining step in the reaction mechanism.
蛋白质法尼基转移酶（FTase）催化Ras以及其他几种参与细胞调控的关键蛋白的异戊烯化。通过预稳态和稳态动力学分析阐明了FTase反应的机制。FTase催化法尼基焦磷酸（FPP）将生物素化肽底物（BiopepSH）法尼基化形成S-法尼基化肽（BiopepS-C15）。稳态动力学机制是有序的。FTase通过两步过程结合FPP，有效解离速率常数为0.013 s-1，总解离常数Kd为2.8 nM。BiopepSH与FTase.FPP不可逆地反应，二级速率常数为2.2×10(5) M-1 s-1，形成FTase.BiopepS-C15。因为在高浓度BiopepSH下，FTase.FPP中的大部分FPP被捕获为FTase.BiopepS-C15，相对于催化作用，FPP从三元复合物中缓慢解离，所以催化的确定性很高。形成FTase.BiopepS-C15（酶结合产物）的最大速率常数远大于kcat（0.06 s-1），表明产物释放是反应机制中的速率决定步骤。
