Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase.
Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase.
Rasmussen
J T
JT
Protein Chemistry Laboratory, University of Aarhus, Denmark. trige@imsb.au.dk
Rasmussen
M S
MS
Petersen
T E
TE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D004226:Dithioerythritol; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism
D013439:Sulfhydryl Reagents; D004226:Dithioerythritol; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D003545:Cysteine; D019807:Iodoacetic Acid