MECHANISM OF INACTIVATION OF NEURONAL NITRIC-OXIDE SYNTHASE BY N-OMEGA-ALLYL-L-ARGININE

N-omega-Allyl-L-arginine is shown to be a competitive reversible inhib itor and time-dependent inactivator of bovine brain nitric oxide synth ase (nNOS). The enzyme is protected against inactivation by the presen ce of the substrate, the absence of O-2, Or if NADP(+) is substituted for NADPH. The NADPH absorption spectrum is converted to that of NADP( +) concomitant with inactivation. The latter two results indicate that redox chemistry is required for inactivation. N-omega-Allyl-N-omega-h ydroxy-L-arginine is synthesized and shown also to be a competitive in hibitor and time-dependent inactivator of nNOS, suggesting that it is a viable intermediate in the inactivation process. Inactivation of nNO S with N-omega-allyl-[C-14]-L-arginine or N-omega-[H-3]allyl-L-arginin e followed by gel filtration or dialysis results in no radioactivity b ound to the enzyme. It is shown spectrophotometrically as well as by H PLC that the heme is modified to four different species during inactiv ation. Inductively coupled plasma atomic emission spectrophotometry is used to show that 1 equiv of ferric ion is present in the modified he mes. When the heme is isolated after inactivation by the two radiolabe led inactivators, it is found that no C-14 is associated, but 0.9 equi v of H-3 is bound to the heme. This indicates that only the allyl part of the inactivator is bound to the heme. HPLC-electrospray mass spect rometry is used to show that the four modified hemes have the same mas s which corresponds to heme plus an allyl group plus a hydrogen. The f act that the modified hemes no longer have an absorption at 400 nm but , instead, absorb at 280 nm suggests that four reduced and allylated h emes are produced (such as 34 or 35). N-omega-Propyl-L-arginine also i s shown to be a competitive inhibitor and time-dependent inactivator o f nNOS; inactivation requires O-2 and NADPH, and the substrate protect s the enzyme from inactivation. Twenty-six equivalents of citrulline a nd nitric oxide is produced during inactivation with N-omega-propyl-L- arginine. This suggests that the double bond of the allyl group is not important to the inactivation mechanism. Possible mechanisms that rat ionalize these results are suggested. No N-omega-allyl-L-citrulline (3 9) is detected from the inactivation of nNOS by N-omega-allyl-L-argini ne, which indicates that initial hydroxylation of the guanidino imine nitrogen does not occur. The only radioactive metabolite generated fro m N-omega-allyl-[C-14]-L-arginine inactivation is citrulline (13 equiv ). Approximately 13 equiv of nitric oxide also are generated. When N-o mega-allyl-N-omega-hydroxy-L-arginine is the inactivator, about 20 equ iv of citrulline and nitric oxide is produced. When N-omega-[H-3]allyl -L-arginine is the inactivator, approximately equal amounts of (H2O)-H -3 and acrolein are produced (8-9 equiv); arginine also is a product. Acrolein does not inactivate nNOS. These last few findings support cle avage of the alpha-C-H bond of the allyl group as a turnover pathway w hich does not lead to inactivation.