Chain cleavage and sulfoxidation of thiastearoyl-ACP upon reaction with stearoyl-ACP desaturase.

The fatty acid analogues 9- and 10-thiastearate were converted to acyl-ACP derivatives by in vitro enzymatic synthesis and reacted with the reconstituted soluble stearoyl-ACP Delta9 desaturase complex. Electrospray ionization mass spectral analysis of the acyl chains purified from the reaction mixtures showed that 10-thiastearoyl-ACP was converted to the 10-sulfoxide as the sole product. In the presence of (18)O(2), the sulfoxide oxygen was found to be derived exclusively from O(2). This result confirms the ability of the soluble stearoyl-ACP desaturase to catalyze O atom transfer in the presence of the appropriate substrate analogue. Inhibition studies showed that 10-thiastearoyl-ACP was a mixed-type inhibitor of 18:0-ACP, with an apparent K(I) of approximately 10 microM. Comparable reactions of the stearoyl-ACP desaturase complex with 9-thiastearoyl-ACP gave the 9-sulfoxide as approximately 5% of the total products, with the O atom again exclusively derived from O(2). The remaining 95% of the total products arose from an acyl chain cleavage reaction between S-9 and C-10. Matrix-assisted laser desorption ionization time-of-flight mass spectral analysis showed that 9-thiastearoyl-ACP had a mass of 9443 amu while the acyl chain cleavage product had a mass of 9322 amu, corresponding to the calculated mass of 8-mercaptooctanoyl-ACP. Recovery of the acyl chain from the ACP product gave the disulfide of 8-mercaptooctanoate (mass of 349.1 amu), arising from the dimerization of 8-mercaptooctanoate during product workup. Gas chromatography-mass spectral analysis also showed the accumulation of nonanal in sealed reaction vials, accounting for the other product of the acyl chain cleavage reaction. The reactivity at both the 9 and 10 positions of the thia-substituted acyl-ACPs is consistent with the proximity of both positions to the diiron center oxidant in the enzyme-substrate complex. Moreover, the differential reactivity of the 9- and 10-thiastearoyl-ACPs also suggests position-dependent consequences of the reaction within the Delta9D active site. Mechanisms accounting for both sulfoxidation and acyl cleavage reactions by the stearoyl-ACP Delta9 desaturase are proposed.