THE OXIDATION OF SELENOCYSTEINE IS INVOLVED IN THE INACTIVATION OF GLUTATHIONE-PEROXIDASE BY NITRIC-OXIDE DONOR

Glutathione peroxidase (GPx) was inactivated by S-nitroso-N-acetyl-D,L -penicillamine (SNAP), a nitric oxide donor (Asahi, M., Fujii, J., Suz uki, K., See, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Ta niguchi, N. (1995) J. Biol. Chem. 270, 21035-21039). The structural ba sis of the inactivation was studied. We also show that 3-morpholinosyd nonimine N-ethylcarbamide, a peroxynitrite precursor, as well as synth etic peroxynitrite also inactivated bovine GPx. The degree of incorpor ation of a sulfhydryl reagent, n-octyldithionitrobenzoic acid, into GP x decreased after pretreatment with SNAP as evidenced by mass spectrom etry. To identify the modification site of this enzyme by SNAP, both S NAP-pretreated and untreated GPxs were reacted with n-octyldithionitro benzoic acid and digested with lysylendopeptidase, and the resulting p eptides were subjected to mass spectrometry. This technique identified a bridge between two peptides, one of which contains Sec(45) at the c atalytic center and Cys(74), and the other contains Cys(91). Although there are two possible combinations, selenocysteine 45 (Sec(45)) and C ys(91) or Cys(74) and Cys(91), the tertiary structure of GPx indicates that a cross-link between Sec(45) and Cys(91) is more feasible. This is consistent with the experimental evidence that SNAP specifically in activates GPx, in which Sec(45) forms the catalytic center. Thus, we c onclude that SNAP mainly oxidized Sec(45) to form a selenenyl sulfide (Se-S) with a free thiol, leading to the inactivation of the enzyme. T hese data suggest that nitric oxide and its derivatives directly inact ivate GPx in a specific manner via the production of a selenenyl sulfi de, resulting in an increase in intracellular peroxides that are respo nsible for cellular damage.