High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

Abstract

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.Keywords: tryptophan fluorescence lifetime; fluorescence quenching; disulphide (disulfide) bridges; photochemical reaction; protein structure damaged by UV light; SS bond disruption; indole; theoretical model Two divergent theories of the mechanisms involved in ultraviolet (UV) inactivation of enzymes have been developed over a period of years. One theory proposes that the random destruction of any amino acid residue causes inactivation (Augenstein and Riley 1964). The second emphasizes the importance of the disruption of a cluster of specific cystine residues (cysteines involved in disulphide bridges; Augenstein and Riley 1964). It was also found that the effective destruction of cystine, tryptophan, tyrosine, and phenylalanine occurs on UV irradiation of proteins (Kazutomo

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