Mechanism-based inactivation of lacrimal-gland peroxidase by phenylhydrazine: a suicidal substrate to probe the active site

Abstract

Humans are exposed to various hydrazine derivatives for therapeutic control of several diseases, and mammalian peroxidases are implicated in the oxidative metabolism of many drugs. The results presented here indicate that lacrimal-gland peroxidase is irreversibly inactivated in a mechanism-based way by phenylhydrazine, which acts as a suicidal substrate in the presence of H₂O₂. The pseudo-first-order kinetic constants for inactivation at pH 5.5 are Ki=18 μM, k_inact=0.25 min^-1 and τ₅₀=2.75 min, with a second-order rate constant of 0.75× 10⁴ 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-dependent inactivation kinetics indicate the involvement of an ionizable group on the enzyme with a pK_a value of 5.4, protonation of which favours 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 μM, and its binding is prevented by 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 formation of a spin-trapped phenyl radical (a^N=15.9G and aβ^H=24.8G) 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, spectral loss is prevented and the enzyme compound II is readily reduced to the native state by phenylhydrazine. The phenylhydrazine-inactivated enzyme reacts with H₂O₂ or CN^- to form compound II or the cyanide complex with a characteristic spectrum, indicating that haem iron is protected from attack by the radical species. The inactivated enzyme binds SCN^- with a K_D value similar to that of the native enzyme (15±3 mM), suggesting that the donor-binding site remains unaffected. CD studies of the inactive enzyme show complete disappearance of the Soret band at 409 nm with the appearance of a new band at 275 nm. This indicates that the haem environment of the enzyme is perturbed in the inactive 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 by incorporation in the vicinity of its haem moiety. The data support the use of phenylhydrazine as a probe for structural and mechanistic analysis of the active site of the lacrimal-gland peroxidase.