Nature of o-phthalaldehyde reaction with pigeon liver fatty acid synthetase.

ABSTRACT

Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation.