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Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.


ABSTRACT: 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 H 2O 2 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 p K 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.

SUBMITTER: Pouwels LJ 

PROVIDER: S-EPMC2643082 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.

Pouwels Lauren J LJ   Zhang Liping L   Chan Nam H NH   Dorrestein Pieter C PC   Wachter Rebekka M RM  

Biochemistry 20080830 38


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. Progr  ...[more]

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