The Efficiency of Enzymatic L-Cysteine Oxidation in Mammalian Systems Derives from the Optimal Organization of the Active Site of Cysteine Dioxygenase
Type of Degreedissertation
Chemistry and Biochemistry
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Intracellular concentrations of free cysteine in mammalian organisms are maintained within a healthy equilibrium by the mononuclear iron-dependent enzyme, cysteine dioxygenase (CDO). CDO catalyzes the oxidation of L-cysteine to L-cysteine sulfinic acid (L-CSA), by incorporating both atoms of molecular oxygen into the thiol group of L-cysteine. The product of this reaction, L-cysteine sulfinic acid, lies at a metabolic branch-point that leads to the formation of pyruvate and sulfate or taurine. The available three-dimensional structures of CDO have revealed the presence of two very interesting features within the active site (1-3). First, in close proximity to the active site iron, is a covalent crosslink between Cys93 and Tyr157. The functions of this structure and the factors that lead to its biogenesis in CDO have been a subject of vigorous investigation. Purified recombinant CDO exists as a mixture of the crosslinked and non crosslinked isoforms, and previous studies of CDO have involved a heterogenous mixture of the two. The current study presents a new method of expressing homogenously non crosslinked CDO using the cell permeative metal chelator, 1,10-phenanthroline. Electron paramagnetic resonance (EPR) analysis of purified non crosslinked CDO revealed that the iron was in the EPR silent Fe2+ form. Dioxygen utilization by non crosslinked CDO occurred in two distinct phases, which correlated with crosslink formation and enzymatic cysteine oxidation. Generation of homogenously crosslinked CDO resulted in a ~5 fold higher kcat/Km value compared to the enzyme with a heterogenous mixture of crosslinked and non crosslinked CDO isoforms. EPR analysis of homogenously crosslinked CDO revealed that this isoform exists in the Fe3+ form. The current studies present a new perspective on the redox properties of the active site iron and the results demonstrate that a redox switch commits CDO towards either formation of the Cys93-Tyr157 crosslink or cysteine oxidation. The second unusual structural feature in the active site of CDO involves the catalytic iron which is ligated by only 3-His residues. The 3-His metal coordination pattern is a deviation from the 3-His/1-Glu metal coordination pattern that is commonly observed in most cupin metalloproteins. A clear rationale behind this deviation is still lacking, but evidence is accumulating that indicate that the deviation might be a mechanistically driven strategy to optimize the dioxygenation reaction. The three-dimensional structure of CDO in complex with L-cysteine revealed a bidentate mode of substrate coordination via the thiol and amino groups. The current studies have evaluated the relevance of the functional groups of L-cysteine in catalysis and the results indicate a remarkable substrate specificity exhibited by CDO. In fact, kinetic analyses of wild-type CDO with structural analogs of L-cysteine revealed a ~30-fold decrease in the kcat/Km value with either D-cysteine or with cysteamine, and there was no detectable activity with 3-mercaptopropionate. Additionally, the relevant enzyme-substrate interactions were evaluated using variants of Tyr58 and Arg60 residues which are in close proximity to the active site iron and have been proposed to play a role in substrate coordination (1-3). The protonation states of key functional groups that are relevant to the catalytic mechanism of CDO were evaluated through determination of the pH dependence of the kinetic parameters of wild-type CDO. Taken together, the results from the current studies have provided invaluable insights into the influence of second sphere interactions on catalysis, and the relevant protonation states of groups participating in catalysis.