Fu H W, Beese L S, Casey P J
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710-3686, USA.
Biochemistry. 1998 Mar 31;37(13):4465-72. doi: 10.1021/bi972511c.
Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the prenylation of several proteins that are important in cellular regulatory events. A specific residue of FTase, Cys299 in the beta subunit previously identified as essential for zinc binding and catalysis, had been tentatively assigned as one of the zinc ligands. This assignment was subsequently confirmed in the X-ray structure of FTase, which also identified two additional residues, Asp297 and His362 in the beta subunit, as the remaining protein-derived metal ligands. To more fully explore the role of zinc in the catalytic mechanism of FTase, site-directed mutagenesis was performed on these two zinc ligands. Although the abilities of all the mutants to bind the farnesyl diphosphate substrate were similar to that of the wild-type enzyme, all the mutants displayed markedly reduced enzymatic activities and zinc affinities. Steady-state and pre-steady-state kinetic analyses of the residual activities indicated that the rate-limiting step changed from product release in the wild-type enzyme to the chemical step of product formation for three of the mutant enzymes. Additionally, single-turnover experiments indicated that the greatest effect of alteration of zinc ligands for all the mutants was on the product formation step, this being reduced 10(3)-10(5)-fold in the mutant forms compared to the wild-type enzyme. These results confirm a critical involvement of the zinc in catalysis by FTase and support a model in which the metal ion is directly involved in the chemical step of the enzymatic reaction.
蛋白质法尼基转移酶（FTase）是一种锌金属酶，可催化几种在细胞调节事件中起重要作用的蛋白质的异戊二烯化。FTase的一个特定残基，即先前确定对锌结合和催化至关重要的β亚基中的Cys299，曾被初步指定为锌配体之一。这一指定随后在FTase的X射线结构中得到证实，该结构还确定了β亚基中的另外两个残基Asp297和His362为其余的蛋白质衍生金属配体。为了更全面地探索锌在FTase催化机制中的作用，对这两个锌配体进行了定点诱变。尽管所有突变体结合法尼基二磷酸底物的能力与野生型酶相似，但所有突变体的酶活性和锌亲和力均显著降低。对残余活性的稳态和预稳态动力学分析表明，对于三种突变酶，限速步骤从野生型酶中的产物释放转变为产物形成的化学步骤。此外，单周转实验表明，所有突变体锌配体改变的最大影响在于产物形成步骤，与野生型酶相比，突变体形式的该步骤减少了10³-10⁵倍。这些结果证实了锌在FTase催化中起关键作用，并支持了一种金属离子直接参与酶促反应化学步骤的模型。
