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Probing the Mechanism of Cysteine Dioxygenase through Substitution of Conserved Active Site Residues
Author(s) -
Imsand Erin,
Ellis Holly R
Publication year - 2008
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.22.1_supplement.611.18
Subject(s) - cysteine , taurine , chemistry , active site , amino acid , site directed mutagenesis , mutagenesis , dioxygenase , biochemistry , binding site , stereochemistry , catalysis , enzyme , mutant , gene
Cysteine dioxygenase (CDO) catalyzes the oxidation of L‐cysteine to L‐cysteine sulfinic acid. This reaction is the rate‐limiting step in the eukaryotic taurine biosynthetic pathway, and taurine levels are directly correlated to cysteine dioxygenase activity. Taurine is an important antioxidant and low levels of taurine have been linked to a variety of cardiac, neural, and autoimmune disorders. Catalytic mechanisms for CDO have been proposed based on recently published three‐dimensional crystal structures, although little direct spectroscopic or kinetic evidence is available to verify these proposals. To probe the mechanism, site‐directed mutagenesis was performed on conserved residues located in the active site of CDO adjacent to the iron center. Substitutions were made for Tyr58, Arg60, Ser153, and His155, replacing each residue with Ala. Kinetic parameters were established and compared to wild‐type to determine if any of the substitutions plays a role in catalysis. Several spectroscopic probes were used to characterize the metal coordination environment. Electron paramagnetic resonance (EPR) spectroscopy in combination with nitric oxide binding studies were performed to simulate the binding of dioxygen with or without the cysteine substrate. Amino acid residues critical to catalysis have been identified in these studies, and a mechanism for cysteine oxidation by CDO is proposed.