Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.
Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.
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DOI：
10.1021/bi8007164
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发表时间：
2008-09-23
期刊：
影响因子：
2.9
通讯作者：
Wachter, Rebekka M.
中科院分区：
文献类型：
--
作者：
Pouwels, Lauren J.;Zhang, Liping;Chan, Nam H.;Dorrestein, Pieter C.;Wachter, Rebekka M.
绿色荧光蛋白（GFP）的成熟过程需要由自发骨架缩合引发的蛋白质氧化反应。发色团是通过 Tyr66 酚基与杂环的完全缀合产生的，该过程需要在苄基碳上发生 C-H 键断裂。我们制备了富含同位素的蛋白质，其β碳上有氘化的酪氨酸残基，并确定了 GFP 自加工反应的动力学同位素效应 (KIE)。通过全局曲线拟合描述氧化前、氧化和氧化后事件的三步机制来分析 H2O2 和成熟发色团产生的进度曲线。尽管无法辨别蛋白质氧化的 KIE (kH/kD = 1.1 ± 0.2)，但在后氧化步骤中提取了 5.9 (± 2.8) 的完整初级 KIE。因此，环外碳不参与分子氧的还原。相反，C-H键断裂是从氧化的环状亚胺形式开始的，并且是最后一步的限速事件。在催化谷氨酸 (E222Q) 取代后观察到成熟的显着 pH 依赖性，表明碱性催化剂的表观 pKa 为 9.4 (± 0.1)。对于该变体，确定了固有时间常数的 KIE 为 5.8 (± 0.4)，该常数被认为描述了最后一步，并得到超高分辨率质谱结果的支持。该数据与产生绿色的后氧化事件的一般碱催化一致。结构论证提出了一种机制，其中高度保守的 Arg96 充当从 Tyr66 衍生的 β 碳中提取质子的催化碱基。
The maturation process of green fluorescent protein (GFP) entails a protein oxidation reaction triggered by spontaneous backbone condensation. The chromophore is generated by full conjugation of the Tyr66 phenolic group with the heterocycle, a process that requires C-H bond scission at the benzylic carbon. We have prepared isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon, and have determined kinetic isotope effects (KIEs) on the GFP self-processing reaction. Progress curves for the production of H2O2 and the mature chromophore were analyzed by global curve fitting to a three-step mechanism describing pre-oxidation, oxidation and post-oxidation events. Although a KIE for protein oxidation could not be discerned (kH/kD = 1.1 ± 0.2), a full primary KIE of 5.9 (± 2.8) was extracted for the post-oxidation step. Therefore, the exocyclic carbon is not involved in the reduction of molecular oxygen. Rather, C-H bond cleavage proceeds from the oxidized cyclic imine form, and is the rate-limiting event of the final step. Substantial pH-dependence of maturation was observed upon substitution of the catalytic glutamate (E222Q), indicating an apparent pKa of 9.4 (± 0.1) for the base catalyst. For this variant, a KIE of 5.8 (± 0.4) was determined for the intrinsic time constant that is thought to describe the final step, as supported by ultra-high resolution mass spectrometric results. The data are consistent with general base catalysis of the post-oxidation events yielding green color. Structural arguments suggest a mechanism in which the highly conserved Arg96 serves as catalytic base in proton abstraction from the Tyr66-derived beta carbon.
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