Glycerol's influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study

Glycerol, employed to mimic biological media with restricted water activity , has been shown to modify the activity of subtilisin BPN', an endopeptidas e, towards the oxidized insulin B-chain, a well-studied substrate(FEBS Lett ., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the stru cture of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain t ertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn r egion of the insulin B-chain; whereas, in glycerol, the peptide is more rig id and is not folded. Moreover, in our experimental conditions, glycerol fa vors beta-strand secondary structure formation. Following these results, hy potheses about the differences observed in enzymatic activity on this subst rate in glycerol have been postulated. (C) 1999 Elsevier Science B.V. All r ights reserved.