Mechanism-Based Inactivation of Lacrimal-Gland Peroxidase by phenylhydrazine : A suicidal substrate to probe the Active Site
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Mechanism-Based Inactivation of Lacrimal-Gland Peroxidase by
phenylhydrazine : A suicidal substrate to probe the Active Site
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| Creator |
Majumder, Avijit
Adak, Subrata Chatterjee, Ratna Banerjee, Ranajit K |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
Humans are exposed to various hydrazine derivatives for therapeuticM control of several diseases, and mammalian peroxidasesN 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 ofH#O#. The pseudo-Ærst-order kinetic constants for inactivation at pH 5±5 are KiØ18 lM, kinactØ0±25 min−" and s&!Ø2±75 min,
with a second-order rate constant of 0±75¨10% 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 pKa 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 diåerence spectroscopy indicate that phenylhydrazine interacts with the enzyme with a KD value of 60 lM, 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 (aNØ15±9 G and abHØ24±8 G) generated on incubation of phenylhydrazine with the enzyme and H#O#. 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 H#O# 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 KD value similar to that of the native enzyme (15³3 mM), suggesting that the donorbinding site remains unaåected. 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 eåect 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.
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| Date |
1997
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/1017/1/Bio_jabhijit2.pdf
Majumder, Avijit and Adak, Subrata and Chatterjee, Ratna and Banerjee, Ranajit K (1997) Mechanism-Based Inactivation of Lacrimal-Gland Peroxidase by phenylhydrazine : A suicidal substrate to probe the Active Site. Biochemical Journal, 324. pp. 713-719. |
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| Relation |
http://dx.doi.org/
http://www.eprints.iicb.res.in/1017/ |
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