3-Carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene: a site-directed inactivator of yeast oxidosqualene cyclase.
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'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-nitro-5-thiocyanobenzoic acid (NTCB), with 3-carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene), a new thiol reagent designed as a site-directed 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 (Ki = 0.67 and 1.21 mM), whereas NTCB was less effective (Ki = 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.
Balliano
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G
Istituto di Chimica Farmaceutica Applicata, Torino, Italy.
Grosa
G
G
Milla
P
P
Viola
F
F
Cattel
L
L
D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D019751:Intramolecular Transferases; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009579:Nitrobenzoates / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013185:Squalene / Q000031:analogs & derivatives* / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D009579:Nitrobenzoates; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; C084457:3-carboxy-4-nitrophenyldithio-1,1',2-trisnorsqualene; D013185:Squalene; D004228:Dithionitrobenzoic Acid; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase; C008658:2-nitro-5-thiocyanobenzoic acid