Spence R A, Hightower K E, Terry K L, Beese L S, Fierke C A, Casey P J
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2000 Nov 14;39(45):13651-9. doi: 10.1021/bi001084r.
Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.
蛋白质法尼基转移酶催化在其C末端含有甲硫氨酸、丝氨酸、谷氨酰胺或丙氨酸的蛋白质底物的脂质修饰。一种密切相关的酶，I型蛋白质香叶基香叶基转移酶，对含有C末端亮氨酸残基的蛋白质底物进行类似的修饰。对β亚基中361位酪氨酸被亮氨酸取代的蛋白质法尼基转移酶突变体的分析得出结论，该酪氨酸残基的侧链在蛋白质底物的识别中起主要作用。然而，在蛋白质法尼基转移酶的晶体结构中，尚未观察到该酪氨酸残基与肽底物之间的相互作用。为了调和这些明显相互矛盾的数据，对哺乳动物蛋白质法尼基转移酶的Y361L变体进行了全面的动力学表征。对Y361L变体的直接结合测量表明，肽底物的结合实际上比野生型酶紧密约40倍。相比之下，I型蛋白质香叶基香叶基转移酶的肽底物结合非常弱。在预稳态动力学分析中发现了差异的原因，该分析表明Y361L变体在单次周转中催化正常肽底物的法尼基化速率与野生型酶相似，但随后的周转被阻止。这些以及其他研究表明，Y361L变体并没有如稳态参数所推断的那样“切换”蛋白质底物特异性；相反，该变体与其正常底物的产物解离严重受损，这种情况导致稳态活性大大受损。
