MECHANISM-BASED INACTIVATION OF LACRIMAL-GLAND PEROXIDASE BY PHENYLHYDRAZINE - A SUICIDAL SUBSTRATE TO PROBE THE ACTIVE-SITE

Humans are exposed to various hydrazine derivatives for therapeutic co ntrol of several diseases, and mammalian peroxidases are implicated in the oxidative metabolism of many drugs. The results presented here in dicate that lacrimal-gland peroxidase is irreversibly inactivated in a mechanism-based way by phenylhydrazine, which acts as a suicidal subs trate in the presence of H2O2. The pseudo-first-order kinetic constant s for inactivation at pH 5.5 are K-i = 18 mu M, k(inact) = 0.25 min(-1 ) and t(50) = 2.75 min, with a second-order rate constant of 0.75 x 10 (4) M(-1)min(-1). Approx. 27 mol of phenylhydrazine and 54 mol of H2O2 are required per mol of enzyme for complete inactivation. The pH-depe ndent inactivation kinetics indicate the involvement of an ionizable g roup on the enzyme with a pK(a) value of 5.4, protonation of which fav ours inactivation. SCN-, the plausible physiological electron donor of the enzyme, protects it from inactivation. Binding studies by optical difference spectroscopy indicate that phenylhydrazine interacts with the enzyme with a K-D value of 60 mu M, and its binding is prevented b y the presence of SCN-. The enzyme is also protected by 5,5-dimethyl-1 -pyrroline N-oxide, a free-radical trap, suggesting the involvement of a radical species in the inactivation. ESR studies indicate the forma tion of a spin-trapped phenyl radical (a(N) = 15.9 G and a beta(H) = 2 4.8 G) generated on incubation of phenylhydrazine with the enzyme and H2O2. A 75% loss of the Soret spectrum is observed when the enzyme is completely inactivated. However, in the presence of the spin trap, spe ctral loss is prevented and the enzyme compound II is readily reduced to the native state by phenylhydrazine. The phenylhydrazine-inactivate d enzyme reacts with H2O2 or CN- to form compound II or the cyanide co mplex with a characteristic spectrum, indicating that haem iron is pro tected from attack by the radical species. The inactivated enzyme bind s SCN- with a K-D value similar to that of the native enzyme (15+/-3 m M), suggesting that the donor-binding site remains unaffected. CD stud ies of the inactive enzyme show complete disappearance of the Soret ba nd at 409 nm with the appearance of a new band at 275 nm. This indicat es that the haem environment of the enzyme is perturbed in the inactiv e form. As benzene, the end product of phenylhydrazine oxidation, has no effect on the enzyme, we suggest that the phenyl radical formed by one-electron oxidation by catalytically active enzyme inactivates it b y incorporation in the vicinity of its haem moiety. The data support t he use of phenylhydrazine as a probe for structural and mechanistic an alysis of the active site of the lacrimal-gland peroxidase.