Role of tryptophan residues of lipoxygenase-1 in activity, structure and stability: chemical modification studies with N-bromosuccinimide

The tryptophan residues of lipoxygenase-1 (LOX1) from soybeans (Glycine max) were modified using N-bromosuccinimide under both native and denaturing conditions. The accessibility of tryptophan residues was pH-dependent. Only one tryptophan residue was accessible at the optimum pH of enzyme activity and with a decrease in pH from 9 to 2, the accessibility increased. Modification of the accessible four tryptophan residues at pH 4.0 under non-denaturing conditions resulted in complete loss of enzyme activity; one tryptophan residue was critical for enzyme activity. Modification of the tryptophan residues did not alter the substrate binding affinity; the presence of the substrate during modification did not alter the extent of modification. Modification of the surface-exposed tryptophans did not affect (i) the conformation or structural integrity but (ii) decreased the stability.