Role of Tryptophan-208 Residue in Cytochrome c Oxidation by Ascorbate Peroxidase From Leishmania Major-Kinetic Studies on Trp208Phe Mutant and Wild Type Enzyme
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Role of Tryptophan-208 Residue in Cytochrome c Oxidation by Ascorbate Peroxidase From Leishmania Major-Kinetic Studies on Trp208Phe Mutant and Wild Type Enzyme
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| Creator |
Yadav, Rajesh K
Dolai, Subhankar Pal, Swati Adak, Subrata |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP)
and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal
tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis. The mutant W208F
enzyme had no effect on both apparent dissociation constant of the enzyme–cytochrome c complex and Km
value for cytochrome c indicating that cytochrome c binding af.nity to the enzyme did not alter after
mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation
without affecting the second order rate constant of compound I formation. Our diode array stopped-.ow
spectral studies showed that the substrate unbound wild type enzyme reacts with H2O2 to form compound I
(compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as
horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from
409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of
compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and
was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin ð-
cation radical. In case of an enzyme–H2O2–ascorbate system, the kinetic for formation and decay of compound
I and II of a mutant enzymewas almost identical to that of a wild type enzyme. Thus, the results of cytochrome
c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for
electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol
oxidation.
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| Date |
2008
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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/1015/1/bba2007.pdf
Yadav, Rajesh K and Dolai, Subhankar and Pal, Swati and Adak, Subrata (2008) Role of Tryptophan-208 Residue in Cytochrome c Oxidation by Ascorbate Peroxidase From Leishmania Major-Kinetic Studies on Trp208Phe Mutant and Wild Type Enzyme. Biochimica et Biophysica Acta, 1784. pp. 863-871. |
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| Relation |
http://dx.doi.org/10.1016/j.bbapap.2008.02.006
http://www.eprints.iicb.res.in/1015/ |
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