Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase.
The heme in lactoperoxidase is attached to the protein by ester bonds between the heme 1- and 5-methyl groups and Glu-375 and Asp-275, respectively. To investigate the cross-linking process, we have examined the D225E, E375D, and D225E/E375D mutants of bovine lactoperoxidase. The heme in the E375D mutant is only partially covalently bound, but exposure to H(2)O(2) results in complete covalent binding and a fully active protein. Digestion of this mutant shows that the heme is primarily bound through its 5-methyl group. Excess H(2)O(2) increases the proportion of the doubly linked species without augmenting enzyme activity. The D225E mutant has little covalently bound heme and a much lower activity, neither of which are significantly increased by the addition of heme and H(2)O(2). The heme is linked to this protein through a single bond to the 1-methyl group. The D225E/E375D mutant has no covalently bound heme and no activity. A small amount of iron 1-hydroxymethylprotoporphyrin IX is obtained from the wild-type enzyme along with the predominant dihydroxylated derivative. The results establish that a single covalent link suffices to achieve maximum catalytic activity and suggest that the 5-hydroxymethyl bond may form before the 1-hydroxymethyl bond.
Colas
Christophe
C
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143-0446, USA.
Kuo
Jane M
JM
Ortiz de Montellano
Paul R
PR
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D018698:Glutamic Acid / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007501:Iron / Q000737:chemistry; D007784:Lactoperoxidase / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011166:Porphyrins / Q000494:pharmacology; D011487:Protein Conformation; D011524:Protoporphyrins / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D011166:Porphyrins; D011524:Protoporphyrins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D006861:Hydrogen Peroxide; C028025:protoporphyrin IX; D007501:Iron; D007784:Lactoperoxidase