Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase.
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 degrees 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.
Mazumdar
Abhijit
A
Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
Bandyopadhyay
Debashis
D
Bandyopadhyay
Uday
U
Banerjee
Ranajit K
RK
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000737:chemistry; D007700:Kinetics; D007765:Lacrimal Apparatus / Q000201:enzymology*; D008956:Models, Chemical; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012756:Sheep; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
C072750:N-carbethoxyhistidine; D019811:Hydroxylamine; D006418:Heme; D006639:Histidine; D009195:Peroxidase; D004047:Diethyl Pyrocarbonate