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Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe site influence the O₂ coupling efficiency in mouse cysteine dioxygenase.
- Source :
-
Biochemistry [Biochemistry] 2013 Dec 23; Vol. 52 (51), pp. 9104-19. Date of Electronic Publication: 2013 Dec 11. - Publication Year :
- 2013
-
Abstract
- Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O₂-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O₂/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O₂/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O₂/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/₂) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.
- Subjects :
- Amino Acid Substitution
Animals
Apoenzymes chemistry
Apoenzymes genetics
Apoenzymes metabolism
Binding Sites
Biocatalysis drug effects
Catalytic Domain
Cysteine analogs & derivatives
Cysteine chemistry
Cysteine metabolism
Cysteine Dioxygenase antagonists & inhibitors
Cysteine Dioxygenase chemistry
Cysteine Dioxygenase genetics
Enzyme Inhibitors pharmacology
Holoenzymes chemistry
Holoenzymes genetics
Holoenzymes metabolism
Kinetics
Mice
Mutant Proteins chemistry
Mutant Proteins metabolism
Oxidation-Reduction drug effects
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Tyrosine chemistry
Tyrosine metabolism
Cysteine Dioxygenase metabolism
Iron metabolism
Models, Molecular
Oxygen metabolism
Protein Processing, Post-Translational
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 52
- Issue :
- 51
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 24279989
- Full Text :
- https://doi.org/10.1021/bi4010232