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3‐carboxy‐4‐nitrophenyl‐dithio‐1,1′,2‐ tris norsqualene: A site‐directed inactivator of yeast oxidosqualene cyclase
Author(s) -
Balliano Gianni,
Grosa Giorgio,
Milla Paola,
Viola Franca,
Cattel Luigi
Publication year - 1993
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02537498
Subject(s) - dtnb , chemistry , substrate (aquarium) , squalene , thiol , reagent , enzyme , biochemistry , dithiothreitol , active site , saccharomyces cerevisiae , stereochemistry , yeast , biology , glutathione , organic chemistry , ecology
The role and location of essential thiol groups in 2,3‐oxidosqualene cyclase from Saccharomyces cerevisiae was examined (i) by comparing inactivation properties of two known thiol reagents, 5,5′‐dithio bis (2‐nitrobenzoic acid) (DTNB) and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB), with 3‐carboxy‐4‐nitrophenyl‐dithio‐1,1′,2‐ tris norsqualene (CNDT‐squalene), a new thiol reagent designed as a sitedirected inactivator of oxidosqualene cyclase and (ii) by testing the ability of the substrate to protect the enzyme against inactivation by the reagents. All reagents gave a time‐dependent inactivation following pseudo‐first order kinetics. DTNB and CNDT‐squalene showed comparable inactivation ability (K i =0.67 and 1.21 mM), whereas NTCB was less effective (K i =15.6 mM). Strong differences between the two most active inhibitors, DTNB and CNDT‐squalene, were observed when the enzyme was saturated with substrate prior to incubation with the thiol reagent. While substrate did not protect the enzyme against the inactivation caused by DTNB, a reduction in the inactivation ability of CNDT‐squalene was observed under protection conditions. The data suggest that the squalene‐like inactivator modifies a thiol group located at the active site of the enzyme.