ARBOXY-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 c yclase from Saccharomyces cerevisiae was examined (i) by comparing ina ctivation properties of two known thiol reagents, 5,5'-dithiobis(2-nit robenzoic acid) (DTNB) and 2-nitro-5-thiocyanobenzoic acid (NTCB), wit h arboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene), a new thiol reagent designed as a site-directed inactivator of oxidosq ualene cyclase and (ii) by testing the ability of the substrate to pro tect the enzyme against inactivation by the reagents. All reagents gav e 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 CN DT-squalene, were observed when the enzyme was saturated with substrat e prior to incubation with the thiol reagent. While substrate did not protect the enzyme against the inactivation caused by DTNB, a reductio n in the inactivation ability of CNDT-squalene was observed under prot ection conditions. The data suggest that the squalene-like inactivator modifies a thiol group located at the active site of the enzyme.