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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 22, Number 1
BKCSDE 22(1)
January 20, 2001 

Identification of an Essential Tryptophan Residue in Alliinase from Garlic (Allium sativum) by Chemical Modification
Young Nam Jin, Yong-Hoon Choi, Chul-Hak Yang
Alliinase active site, Tryptophan of alliinase.
We have employed chemical modification to identify amino acids essential for the catalytic activity of alliinase (EC from garlic (Allium sativum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the characteristic odor of garlic. The activity of alliinase was rapidly and completely inactivated by N-bromosuccinimide(NBS) and slightly decreased by succinic anhydride and N-acetylimidazole. These results indicate that tryptophanyl, lysyl, and tyrosyl residues play an important role in enzyme catalysis. The reaction of alliinase with NBA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with increasing reagent concentration of NBS, consistent with the oxidation of tryptophan residues. Kinetic analysis, fluorometric titration of tryptophans and correlation to residual alliinase activity showed that modification of only one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemical modification by NBS in the presence and absence of the protecting substrate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing residues 179-188 was isolated. We conclude that Trp182 is essential for alliinase activity.
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