Pickett Jennifer S, Bowers Katherine E, Hartman Heather L, Fu Hua-Wen, Embry Alan C, Casey Patrick J, Fierke Carol A
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Biochemistry. 2003 Aug 19;42(32):9741-8. doi: 10.1021/bi0346852.
The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from farnesyl diphosphate (FPP) to a cysteine residue near the C-terminus of a protein substrate. Several crystal structures of inactive FTase.FPP.peptide complexes indicate that K164alpha interacts with the alpha-phosphate and that H248beta and Y300beta form hydrogen bonds with the beta-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601-16611]. Mutations K164Aalpha, H248Abeta, and Y300Fbeta were prepared and analyzed by single turnover kinetics and ligand binding studies. These mutations do not significantly affect the enzyme affinity for FPP but do decrease the farnesylation rate constant by 30-, 10-, and 500-fold, respectively. These mutations have little effect on the pH and magnesium dependence of the farnesylation rate constant, demonstrating that the side chains of K164alpha, Y300beta, and H248beta do not function either as general acid-base catalysts or as magnesium ligands. Mutation of H248beta and Y300beta, but not K164alpha, decreases the farnesylation rate constant using farnesyl monophosphate (FMP). These data suggest that, contrary to the conclusions derived from analysis of the static crystal structures, the transition state for farnesylation is stabilized by interactions between the alpha-phosphate of the isoprenoid substrate and the side chains of Y300beta and H248beta. These results suggest an active substrate conformation for FTase wherein the C1 carbon of the FPP substrate moves toward the zinc-bound thiolate of the protein substrate to react, resulting in a rearrangement of the diphosphate group relative to its ground state position in the binding pocket.
锌金属酶蛋白法尼基转移酶（FTase）催化法尼基二磷酸（FPP）上15碳的法尼基部分转移至蛋白质底物C末端附近的半胱氨酸残基上。无活性的FTase.FPP.肽复合物的几个晶体结构表明，K164α与α-磷酸相互作用，H248β和Y300β与FPP的β-磷酸形成氢键[斯特里克兰，C.L.等人（1998年）《生物化学》37卷，16601 - 16611页]。制备了K164Aα、H248Aβ和Y300Fβ突变体，并通过单周转动力学和配体结合研究进行分析。这些突变对酶与FPP的亲和力没有显著影响，但分别使法尼基化速率常数降低了30倍、10倍和500倍。这些突变对法尼基化速率常数的pH和镁依赖性影响很小，表明K164α、Y300β和H248β的侧链既不作为一般酸碱催化剂，也不作为镁配体发挥作用。H248β和Y300β的突变（而非K164α的突变）降低了使用法尼基单磷酸（FMP）时的法尼基化速率常数。这些数据表明，与从静态晶体结构分析得出的结论相反，法尼基化的过渡态通过类异戊二烯底物的α-磷酸与Y300β和H248β的侧链之间的相互作用得以稳定。这些结果表明了FTase的一种活性底物构象，其中FPP底物的C1碳向蛋白质底物与锌结合的硫醇盐移动以发生反应，导致二磷酸基团相对于其在结合口袋中的基态位置发生重排。
