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

Lauren J. Pouwels, Liping Zhang, Nam H. Chan, Pieter C. Dorrestein, Rebekka Wachter

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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 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 (kH/kD = 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 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 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.

Original languageEnglish (US)
Pages (from-to)10111-10122
Number of pages12
JournalBiochemistry
Volume47
Issue number38
DOIs
StatePublished - Sep 23 2008

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Chromophores
Green Fluorescent Proteins
Isotopes
Carbon
Kinetics
Oxidation
Bearings (structural)
Proteins
Imines
Molecular oxygen
Curve fitting
Catalysis
Tyrosine
Protons
Glutamic Acid
Condensation
Substitution reactions
Color
Oxygen
Catalysts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants. / Pouwels, Lauren J.; Zhang, Liping; Chan, Nam H.; Dorrestein, Pieter C.; Wachter, Rebekka.

In: Biochemistry, Vol. 47, No. 38, 23.09.2008, p. 10111-10122.

Research output: Contribution to journalArticle

Pouwels, Lauren J. ; Zhang, Liping ; Chan, Nam H. ; Dorrestein, Pieter C. ; Wachter, Rebekka. / Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants. In: Biochemistry. 2008 ; Vol. 47, No. 38. pp. 10111-10122.
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