Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum).
Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5'-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S--S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored --SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two --SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free --SH groups. This allowed the oriented conjugation of alliinase, via the --SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
Weiner
Lev
L
Department of Chemical Research Support, The Weizmann Institute of Science, 76100 Rehovot, Israel.
Shin
Irina
I
Shimon
Linda J W
LJ
Miron
Talia
T
Wilchek
Meir
M
Mirelman
David
D
Frolow
Felix
F
Rabinkov
Aharon
A
D001710:Biotin / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005737:Garlic / Q000201:enzymology*; D055767:Immobilized Proteins / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D008301:Maleimides / Q000378:metabolism; D008958:Models, Molecular; D017386:Sequence Homology, Amino Acid; D019809:Streptavidin / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D055767:Immobilized Proteins; D007202:Indicators and Reagents; D008301:Maleimides; D013438:Sulfhydryl Compounds; C016399:N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; D001710:Biotin; D019809:Streptavidin; D013437:Carbon-Sulfur Lyases; C019243:alliin lyase