Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo.
The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.
Oxvig
C
C
Department of Molecular and Structural Biology, Science Park Division, University of Aarhus, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark. co@mbio.aau.dk
Thomsen
A R
AR
Overgaard
M T
MT
Sorensen
E S
ES
Højrup
P
P
Bjerrum
M J
MJ
Gleich
G J
GJ
Sottrup-Jensen
L
L
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology*; D004952:Esters; D018698:Glutamic Acid / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007784:Lactoperoxidase / Q000737:chemistry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D017421:Sequence Analysis
D004952:Esters; D009842:Oligopeptides; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D047209:Eosinophil Peroxidase; D007784:Lactoperoxidase; D010544:Peroxidases