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Genuine Garlic:

Alliinase from Allium sativum

Plants of the genus Allium gain their characteristic taste and odor from a bunch of volatile compounds containing several sulfur atoms each. A prominent role in both traditional medicine and interhuman relationships accounts to garlic (not only in transsylvania). The key enzymatic reaction preceding the generation of the smells is the production of a chemically reactive sulfenic acid which then spontaneously reacts to form all these interesting substances. Alliinase is the enzyme involved, with full name S-alk(en)yl-L-cysteine sulfoxide lyase. The garlic enzyme cleaves S-allyl-L-cysteine sulfoxide in a beta-elimination-deamination reaction involving an aminoacryl intermediate bound to the cofactor pyridoxal-5'-phosphate.

The substrate is bound in the enzyme near to the cofactor to form a Schiff's base.

Enzyme-provided acidic and basic groups induce a polarization of the substrate's S=O bond leading to a rearrangement of bonding electrons. The carbon-sulfur bond is cleaved to leave an acrylamide bound to the cofactor and set the reaction's product (allyl sulfenic acid) free. The Schiff's base is hydrolyzed, and the second product of the reaction decomposes to pyruvate and ammonia.

Garlic alliinase is a dimer of identical subunits of 448 amino acids each. In each monomer a central domain is preceded by a N-terminal domain and followed by a C-terminal domain  .

The aminoterminal domain contains a subdomain resembling an epidermal growth factor  . This motif is characterized by three disulfide bridges  . In animals this structure is used to facilitate binding to other proteins. The aminoterminal domain also contains the binding site for a chloride ion  . The chloride is hydrogen-bonded to the amide nitrogens of Phe94, Ser98, Phe100, and a water molecule. The loop is stabilized by these interactions, and so is the cis-peptide bond of Asn95 to Pro96  .

The central domain contains a seven-stranded sheet flanked by helices which corresponds to other C-S lyases. This domain binds the cofactor pyridoxal phosphate  .

The carboxyterminal domain is structured like a typical aminotransferase: the central sheet is situated against helices  .

Garlic alliinase is a glycoprotein. One of the four sugar chains composed of fucose, N-acetylglucosamine, and mannose was sufficiently ordered in the protein crystal to be completely modelled  . This sugar chain is placed at the dimer interface and contacts both subunits  .

The active site of the enzyme harbours the cofactor pyridoxal phosphate  . It forms a Schiff's base to Lys251  . Besides this covalent bond it is held by numerous other interactions: the pyridine ring is sandwiched between Tyr165 and the sidechain of Val227  , giving pi electron interaction. Hydrogen bonds are formed from the hydroxyl group to Asn207 and Tyr228 and from the pyridine nitrogen to Asp225  . The phosphate group is faced by the positively charged dipole-end of an alpha-helix and the guanidino group of Arg259  . Additionally there are hydrogen bonds to Thr133, Thr248, Ser250, and Tyr92 (the latter being from the other subunit). This tight grip on the cofactor is neccessary, as the covalent bond to Lys251 is broken in the reaction cycle, when the Schiff's base is formed to the substrate.

S-ethyl-L-cysteine lacks a polarizable S=O bond and therefore is an inhibotor for alliinase.

When added in the crystallization experiment, beneath the pyridoxal phosphate an acrylamide residue (reaction product) is found bound instead of the lysine. This gives a hint to the placement of the substrate in the reaction center  .

Restart this demonstration

EB Kuettner et al, The active principle of garlic at atomic resolution, J. Biol. Chem. 277 (2002) 46402-46407

5-03 - R Bergmann