Identification of an essential tryptophan residue in alliinase from garlic(Allium sativum) by chemical modification

We have employed chemical modification to identify amino acids essential fo r the catalytic activity of alliinase (EC 4.4.1.4) from garlic (Allium sati vum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the charact eristic odor of garlic. The activity of alliinase was rapidly and completel y inactivated by N-bromosuccinimide (NBS) and slightly decreased by succini c anhydride and N-acetylimidazole. These results indicate that tryptophanyl , lysyl, and tyrosyl residues play an important role in enzyme catalysis. T he reaction of alliinase with NEA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with inc reasing reagent concentration of NBS, consistent with the oxidation of tryp tophan residues. Kinetic analysis, fluorometric titration of tryptophans an d correlation to residual alliinase activity showed that modification of on ly one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemi cal modification by NBS in the presence and absence of the protecting subst rate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing res idues 179- 186 was isolated. We conclude that Trp182 is essential for allii nase activity.