Low Catalytic Turnover of Horseradish Peroxidase in Thiocyanate Oxidation
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Low Catalytic Turnover of Horseradish Peroxidase in
Thiocyanate Oxidation
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| Creator |
Adak, Subrata
Majumder, Avijit Banerjee, Ranajit K |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
The catalytic turnover of horseradish peroxidase
(HRP) to oxidize SCN2 is a hundredfold lower than that
of lactoperoxidase (LPO) at optimum pH. While studying
the mechanism, HRP was found to be reversibly inactivated
following pseudo-first order kinetics with a second
order rate constant of 400 M21 min21 when incubated
with SCN2 and H2O2. The slow rate of SCN2
oxidation is increased severalfold in the presence of free
radical traps, 5–5-dimethyl-1-pyrroline N-oxide or
a-phenyl-tert-butylnitrone, suggesting the plausible role
of free radical or radical-derived product in the inactivation.
Spectral studies indicate that SCN2 at a lower
concentrations slowly reduces compound II to native
state by one-electron transfer as evidenced by a time-dependent
spectral shift from 418 to 402 nm through an
isosbestic point at 408 nm. In the presence of higher
concentrations of SCN2, a new stable Soret peak appears
at 421 nm with a visible peak at 540 nm, which are
the characteristics of the inactivated enzyme. The oneelectron
oxidation product of SCN2 was identified by
electron spin resonance spectroscopy as 5–5-dimethyl-1-
pyrroline N-oxide adduct of the sulfur-centered thiocyanate
radical (aN 5 15.0 G and abH 5 16.5 G). The inactivation
of the enzyme in the presence of SCN2 and H2O2
is prevented by electron donors such as iodide or guaiacol.
Binding studies indicate that both iodide and guaiacol
compete with SCN2 for binding at or near the SCN2
binding site and thus prevent inactivation. The spectral
characteristics of the inactivated enzyme are exactly
similar to those of the native HRP-CN2 complex. Quantitative
measurements indicate that HRP produces a
10-fold higher amount of CN2 than LPO when incubated
with SCN2 and H2O2. As HRP has higher affinity for CN2
than LPO, it is concurrently inactivated by CN2 formed
during SCN2 oxidation, which is not observed in case of
LPO. This study further reveals that HRP catalyzes
SCN2 oxidation by two one-electron transfers with the
intermediate formation of thiocyanate radicals. The
radicals dimerize to form thiocyanogen, (SCN)2, which
is hydrolyzed to form CN2. As LPO forms OSCN2 as the
major stable oxidation product through a two-electron
transfer mechanism, it is not significantly inactivated
by CN2 formed in a small quantity
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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/1057/1/JBC%2Dthiocyanate.pdf
Adak, Subrata and Majumder, Avijit and Banerjee, Ranajit K (1997) Low Catalytic Turnover of Horseradish Peroxidase in Thiocyanate Oxidation. The Journal of Biological Chemistry, 272 (17). pp. 11049-11056. |
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| Relation |
http://dx.doi.org/
http://www.eprints.iicb.res.in/1057/ |
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