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 H # O # . The pseudo-first-order kinetic constants for inactivation at pH 5n5 are K i l 18 µM, k inact l 0n25 min −" and τ &! l 2n75 min, with a second-order rate constant of 0n75i10% M −" :min −" . Approx. 27 mol of phenylhydrazine and 54 mol of H # O # are required per mol of enzyme for complete inactivation. The pHdependent inactivation kinetics indicate the involvement of an ionizable group on the enzyme with a pK a value of 5n4, 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 l 15n9 G and

Suicide-peroxide inactivation of horseradish peroxidase in the presence ofSodium n-Dodecyl Sulphate: A study of the enzyme deactivation kinetics

“…On the other hand, it is deemed that HRP may go through an additional kinetic mechanism of inactivation namely "suicide-substrate inactivation" besides its two described major catalytic pathways [5,69,73,87,88]. So, the principal and central role of the compound I-peroxide complex is well illustrated in suicide substrate inactivation of the enzyme.…”

Thyroidal and Extrathyroidal Requirements for Iodine and Selenium: A Combined Evolutionary and (Patho)Physiological Approach

“…As said, the peroxidase partner system aims at the destruction of microbes, such as viruses, bacteria and fungi [ 4 , 84 , 86 , 88 , 89 , 90 , 91 , 92 ]. In the extrathyroidal tissues [ 82 ], the system also makes use of other substrates that are transported by the NIS, and are usually considered ‘goitrogens’, like thiocyanate (SCN − ), bromide (Br − ), chloride (Cl − ; see below) and possibly selenocyanate (SeCN − ).…”