Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase.
In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.
Suriano
G
G
Dipartimento di Chimica I. F. M., Universitá di Torino, 7 Via Pietro Giuria, 10125 Torino, Italy.
Watanabe
S
S
Ghibaudi
E M
EM
Bollen
A
A
Ferrari
R P
RP
Moguilevsky
N
N
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018698:Glutamic Acid / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D007784:Lactoperoxidase / Q000737:chemistry*; D009154:Mutation; D014633:Valine / Q000737:chemistry*
D017931:DNA Primers; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D007784:Lactoperoxidase; D014633:Valine; D005998:Glycine