Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase

• Published in: FEBS letters 1999-08, Vol.456 (3), p.409-416
• Main Authors: Prompers, Jeanine J., Hilbers, Cornelis W., Pepermans, Henri A.M.
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Amides - chemistry; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - radiation effects; CSA, chemical shift anisotropy; Cutinase; Cysteine - chemistry; Disulfide bond; Disulfides - chemistry; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); FHSQC, fast HSQC; Fluorescence; Fluorescence spectroscopy; Fusarium - enzymology; HSQC, heteronuclear single-quantum coherence spectroscopy; Hydrogen Bonding; Magnetic Resonance Spectroscopy - methods; Nitrogen Isotopes; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; Nuclear magnetic resonance; Sulfhydryl Compounds - chemistry; Sulfhydryl Reagents - chemistry; Thiol; TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation; Tryptophan - chemistry; Tryptophan - radiation effects; Tryptophan mediated photoreduction
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)00990-4

The fluorescence signal of the single tryptophan residue (Trp 69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp 69 in cutinase is caused by the breaking of the disulfide bond between Cys 31 and Cys 109 upon irradiation, while the amide-aromatic hydrogen bond between Ala 32 and Trp 69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.