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Evaluating the Mechanistic Roles of Amino Acid Residues in the Active Site of Cysteine Dioxygenase
Author(s) -
Graham Claire,
Ellis Holly
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.22
Subject(s) - cysteine , histidine , chemistry , serine , active site , residue (chemistry) , wild type , stereochemistry , tyrosine , amino acid , dioxygenase , enzyme , biochemistry , mutant , gene
Cysteine dioxygenase (CDO) is a mononuclear iron‐containing enzyme that catalyzes the oxidation of L‐cysteine. In the active site of CDO the iron is coordinated by three histidine residues. Neighboring the iron center is a unique thioether crosslink that exists between a tyrosine and cysteine residue. The crosslink has been shown to enhance activity and stabilize the iron center. 1 In addition, Tyr157 interacts with histidine (His155) and serine (Ser153) residues resulting in a hydrogen bonding network. Two variants (H155A and S153A CDO) were constructed to investigate the role of these residues in crosslink formation or cysteine oxidation. There was no difference in the amount of iron bound for each variant compared to wild‐type CDO, suggesting that the hydrogen‐bonding network is not involved in stabilization of the metal center. Cysteine oxidation was not affected by these substitutions, as the steady‐state kinetic parameters for S153A and H155A CDO were analogous to wild type CDO. The H155A and S153A CDO variants were heterogeneously crosslinked as previously observed for the wild type enzyme. However, the H155A CDO variant existed primarily in the noncrosslinked isoform. It was previously shown that the homogeneously crosslinked isoform could be generated in the presence of iron, dioxygen, and L‐cysteine. Interestingly, S153A and wild type CDO were able to form the fully crosslinked species, but the H155A CDO variant remained predominately noncrosslinked. The results from these studies suggest that His155 plays a role in crosslink formation, but is not directly involved in catalytic events involving cysteine oxidation.