Biophysical Chemistry zyxwvutsrqpon ELSEVIER Biophysical Chemistry 58 (1996) 28 l-288 Photophysics of the single tryptophan residue in zyxwvutsrqponmlkjihgfedcbaZYX Fusarium solani Cutinase: Evidence for the occurrence of conformational substates with unusual fluorescence behaviour Petra C.M. Weisenbom a* *, Hans Meder a, Maarten R. Egmond ‘, Ton J.W.G. Visser b, Arie van Hoek b a Unileuer Research Luhoratorium, Olruier ULO? Noortlam 120. 3133 A7 Vluurdingm. Netherlantl.\ b Department of Biochemistry, Agricultural Unioersity. Dreijenluun 3. 6703 HA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM Wqenin~en. Netherlmdt Received 20 February 1995; revised 30 May 1995; accepted 6 June 1995 Abstract The single tryptophan residue, at position 69 in the amino acid sequence, was used as an intrinsic probe to obtain structural and dynamical information on the lipolytic enzyme Fusarium solani cutinase. In the enzyme’s native state the tryptophan fluorescence is highly quenched. Time-resolved experiments reveal that the majority of the excited state snecies is characterized by an unusually fast decay time of approximately 40 ps, indicating the occurrence of a very efficient nonradiative relaxation process, possibly via the adjacent disulphide bond or via the peptide bonds of a nearby loop. A minority of the excited state species relaxes on a nanosecond time scale. Irradiation of the enzyme in the tryptophan absorption band causes an increase by an order of magnitude of the fluorescence quantum yield. This increase is ascribed to a photo-induced, subtle structural change of a minor subset of species whose fluorescence is not highly quenched. The structural change is accompanied by a tightening of the local environment of the tryptophan moiety, as indicated by results from time-resolved fluorescence anisotropy which reveal a complete disappearance of the segmental flexibility of the tryptophan moiety. Keywords: Cutinase; Lipase; Fluorescence; Conformation 1. Introduction Lipolytic enzymes catalyze the hydrolysis of fatty esters such as triacylglycerols. Their activity on tri- acylglycerols is often greatly enhanced at lipid-water * Corresponding author. interfaces. One of the industrial applications of lipolytic enzymes involves their addition to detergent products for fabric washing in order to improve the product’s effectiveness in the removal of fat stains. It is realized, however, that the conditions in a typical detergent solution (such as pH, ionic strength. surfac- tant micelles) generally do not provide an optimal environment for an enzyme to be fully active or even to retain its native conformation. Much research is therefore devoted to fully understand the effects of 030 I -4622/96/$15.00 0 1996 Elsevier zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Science B.V. All rights reserved SD/ 0301.4622(95)00079-8