Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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Title |
Probing the role of active site histidine residues in
the catalytic activity of lacrimal gland peroxidase
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Creator |
Mazumdar, Abhijit
Bandyopadhyay, Debashis Bandyopadhyay, Uday Banerjee, Ranajit K |
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Subject |
Cell Biology & Physiology
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Description |
The role of active site histidine residues in SCN– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification
with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a
second order rate constant of 0.26 M–1 sec–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6
indicating histidine derivatization. The UV difference spectrum of the modified vs. native enzyme shows a peak at 242 nm
indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine
indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show
that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with
SCN– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically
detectable compound II formation is lost due to modification and is not evident after SCN– protection. The data indicate that
out of two histidines, one regulates compound I formation while the other one controls SCN– binding. SCN– protected enzyme
is inactive due to loss of compound I formation. SCN– binding studies by optical difference spectroscopy indicate that while
the native enzyme binds SCN– with the Kd of 15 mM, the modified enzyme shows very weak binding with the Kd of 660 mM.
From the pH dependent binding of SCN–, a plot of log Kd vs. pH shows a sigmoidal curve from which the involvement of an
enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN– binding. LGP has
thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls
SCN- binding. (Mol Cell Biochem 237: 21–30, 2002)
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Publisher |
Kluwer
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Date |
2002
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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/773/1/MOLECULAR_AND_CELLULAR_BIOCHEMISTRY__237(_1%2D2)_21%2D30;2002[36].pdf
Mazumdar, Abhijit and Bandyopadhyay, Debashis and Bandyopadhyay, Uday and Banerjee, Ranajit K (2002) Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase. Molecular and Cellular Biochemistry, 237 (1-2). pp. 21-30. |
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Relation |
http://dx.doi.org/10.1023/A:1016540303300
http://www.eprints.iicb.res.in/773/ |
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