Tobin Daniel A, Pickett Jennifer S, Hartman Heather L, Fierke Carol A, Penner-Hahn James E
Department of Chemistry and Biophysics Research Division, The University of Michigan, Ann Arbor, MH 48109-1055, USA.
J Am Chem Soc. 2003 Aug 20;125(33):9962-9. doi: 10.1021/ja035927o.
X-ray absorption spectroscopy has been used to determine the structure of the Zn site in protein farnesyltransferase. Extended X-ray absorption fine structure (EXAFS) data are consistent with a Zn site that is ligated to three low-Z (oxygen or nitrogen) ligands and one cysteine sulfur, as predicted from the crystal structures that are available for farnesyltransferase. However, in contrast with the crystallographic results the EXAFS data do not show evidence for significant distortions in the Zn-ligand distances. The average Zn-(N/O) and Zn-S distances are 2.04 and 2.31 A, respectively. Addition of a farnesyl diphosphate analogue causes no detectable change in the structure of the Zn site. However, addition of peptide substrate causes a change in ligation from ZnS(N/O)(3) to ZnS(2)(N/O)(2), consistent with ligation of the C-terminal cysteine to the Zn. There is no significant change in Zn-ligand distances when a substrate binds, demonstrating that the Zn remains four-coordinate. Addition of both peptide and farnesyl diphosphate to give the product complex causes the Zn to return to ZnS(N/O)(3) ligation, indicating that the product thioether is not tightly coordinated to the Zn. These spectroscopic experiments provide insight into the catalytic mechanism of FTase.
X射线吸收光谱法已被用于确定蛋白质法尼基转移酶中锌位点的结构。扩展X射线吸收精细结构（EXAFS）数据与一个锌位点相符，该位点与三个低原子序数（氧或氮）配体和一个半胱氨酸硫原子配位，这与法尼基转移酶现有的晶体结构预测一致。然而，与晶体学结果相反，EXAFS数据没有显示锌-配体距离存在明显扭曲的证据。锌-（氮/氧）和锌-硫的平均距离分别为2.04 Å和2.31 Å。添加法尼基二磷酸类似物不会使锌位点的结构发生可检测到的变化。然而，添加肽底物会导致配位从ZnS（氮/氧）₃变为ZnS₂（氮/氧）₂，这与C端半胱氨酸与锌的配位一致。当底物结合时，锌-配体距离没有显著变化，表明锌仍然是四配位的。同时添加肽和法尼基二磷酸以形成产物复合物会使锌回到ZnS（氮/氧）₃配位，这表明产物硫醚与锌的配位不紧密。这些光谱实验为法尼基转移酶的催化机制提供了深入了解。
