Kinetic Isotope Effect Studies on the de Novo Rate of Chromophore Formation in Fast- and Slow-Maturing GFP Variants

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• 作者：Lauren J. Pouwels ; Liping Zhang ; Nam H. Chan ; Pieter C. Dorrestein ; Rebekka M. Wachter
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：September 23, 2008
• 年：2008
• 卷：47
• 期：38
• 页码：10111-10122
• 全文大小：329K
• 年卷期：v.47,no.38(September 23, 2008)
• ISSN：1520-4995

文摘

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 preoxidation, oxidation and postoxidation events. Although a KIE for protein oxidation could not be discerned (k_H/k_D = 1.1 ± 0.2), a full primary KIE of 5.9 (±2.8) was extracted for the postoxidation 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 pK_a 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 postoxidation 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.