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Thiol Dioxygenases: An Investigation Into The Mechanisms Of Cysteine Dioxygenase And 3-mercaptopropionic Acid Dioxygenase
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Thiol dioxygenase (TDO) enzymes catalyze the molecular oxygen-dependent oxidation of sulfur containing amino acid derivatives. The loss of the ability to regulate thiols has been linked to a number of neurological diseases. Two TDO enzymes, a mammalian cysteine dioxygenase (CDO) and a bacterial 3-mercaptopropionic acid dioxygenase, are examined by probing the various effects on the steady-state kinetic parameters kcat and kcat/KM. Both of these enzymes show a similar deviation from the iconic 2-His-1-carboxylate facial triad active site motif that dominates the mononuclear non-heme iron oxidase/oxygenase class of enzymes. However, CDO shows an unusual covalently crosslinked Cys-Tyr pair within 3.3 Å of the active site that is not present in 3-mercaptopropionic acid dioxygenase (MDO). In this work, second-sphere interactions of CDO are probed by observing perturbations to steady-state parameters in the presence of selected active site variants. In addition, the relative timing of chemical and non-chemical steps in both CDO and MDO are investigated by a series of solvent kinetic isotope effects and viscosity studies. These experiments reveal a proton dependent intermediate gates coupling efficiency in CDO. Substrate-enzyme interactions for MDO with three substrates (cysteine (cys), cysteamine (cyst), and 3-mercaptopropionic acid (3mpa)) are investigated by observing steady-state kinetic parameters as a function of pH. Complementary X-band EPR studies were performed using nitric oxide as a surrogate for O2-binding. As with most non-heme mononuclear iron enzymes, obligate-ordered addition of substrate prior to NO is observed. Two distinct substrate-bound conformations were observed in enzyme-substrate-NO samples prepared with either cysteine or cysteamine, suggesting heterogeneous binding of these substrates within the active site. Kinetic and EPR results are consistent with 3mpa being the preferred substrate for this enzyme.