Hightower K E, Huang C C, Casey P J, Fierke C A
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 1998 Nov 3;37(44):15555-62. doi: 10.1021/bi981525v.
The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the microM range (KD = 4 microM) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pKa values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pKa of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pKa of the thiol resulting in formation of a bound thiolate at physiological pH.
锌金属酶蛋白法尼基转移酶（FTase）催化蛋白质底物的半胱氨酸残基与含15个碳原子的法尼基基团发生烷基化反应。我们开发了荧光测定法来直接测量该酶对肽和蛋白质（Ras）底物的亲和力。在生理pH值下，与H-Ras羧基末端对应的肽在微摩尔范围内（KD = 4微摩尔）与FTase结合；然而，在与酶结合的法尼基二磷酸（FPP）类似物形成的三元复合物中，肽的亲和力提高了约70倍，这表明两种底物协同结合。还研究了底物结合的pH依赖性，观察到两个电离过程：对于三元复合物，pKa值分别为8.1（反映游离肽中硫醇的电离）和6.4。钴离子取代的FTase三元复合物的光吸收光谱中配体-金属电荷转移带的pH依赖性表明，与金属配位的硫醇以6.3的pKa发生电离。这些数据表明，FTase中肽硫与锌离子的金属配位降低了硫醇的pKa，导致在生理pH值下形成结合的硫醇盐。
