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THIOL DIOXYGENASES: AN INVESTIGATION INTO THE MECHANISMS OF CYSTEINE DIOXYGENASE AND 3-MERCAPTOPROPIONIC ACID DIOXYGENASE by JOSHUA CROWELL Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON December 2015 Copyright © by Joshua Crowell 2015 All Rights Reserved ii Acknowledgements I would like to thank Dr. Brad S. Pierce for your guidance over the past five years. Your patience and professionalism is deserving of my upmost respect and I will forever appreciate this as an example of how to treat the people I work with. I would like to thank my committee members Dr. Heo and Dr. Jonhson-Winters for their direction and advice. I also would like to thank all my lab mates, Wei, Bishnu, Andra, Priyanka, Eleanor, Sinjinee, Phil, and Mike. It has been a pleasure working with all of you. I would like to thank the Department of Chemistry and Biochemistry for the help with my academic and career. Lastly, I would like to thank my family. Mom and Dad, thank you for the encouragement you have shown my whole life. To my wife, Laura, thank you for your patience and support. Sawyer, Canaan, and Emmett, thank you for making me forget about work every day when I get home. I love you and most certainly couldn’t have done this without any of you. Most importantly, I want to thank God for this amazing opportunity. October 13, 2015 iii Abstract THIOL DIOXYGENASES: AN INVESTIGATION INTO THE MECHANISMS OF CYSTEINE DIOXYGENASE AND 3-MERCAPTOPROPIONIC ACID DIOXYGENASE Joshua Crowell, PhD The University of Texas at Arlington, 2015 Supervising Professor: Brad Pierce 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 cross-linked 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 iv (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. v Table of Contents Acknowledgements ............................................................................................................. iii Abstract .............................................................................................................................. iv List of Illustrations ............................................................................................................... x List of Tables ...................................................................................................................... xii List of Schemes ................................................................................................................. xiii Chapter 1 Introduction to Mononuclear Non-Heme Iron Enzymes ..................................... 1 Enzymes Containing the 2-His 1-Carboxylate Facial Triad ............................................ 2 α-Ketogluterate-Dependent Enzymes ........................................................................ 4 Rieske Dioxygenases ................................................................................................. 6 Catechol Cleaving Dioxygenases ............................................................................... 8 Extradiol dioxygenases .......................................................................................... 9 Intradiol dioxygenase ........................................................................................... 11 Pterin-Dependent Hydroxylases ............................................................................... 12 Other Enzymes ......................................................................................................... 15 ACCO ................................................................................................................... 15 Isopenicillin N synthase ....................................................................................... 16 The 3-His Facial Triad .................................................................................................. 18 Cysteine Dioxygenase .............................................................................................. 19 3-Mercaptopropionic Acid Dioxygenase ................................................................... 27 Chapter 2 Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe-site influence O2-coupling efficiency in mouse cysteine dioxygenase ........................................................................................................ 28 Introduction ................................................................................................................... 28 Materials and Methods ................................................................................................. 34 vi Purification ................................................................................................................ 34 Conversion of as-isolated CDO (AI-CDO) to fully modified CDO (- CDO)......................................................................................................................... 35 HPLC CDO Activity Assay ........................................................................................ 35 Oxygen Electrode ..................................................................................................... 36 pH profile results ....................................................................................................... 37 Analysis of kinetic data ............................................................................................. 37 Spectroscopy ............................................................................................................ 37 Computational Methods............................................................................................ 39 Results .......................................................................................................................... 41 Purification of CDO enzyme forms and selected variants ........................................ 41 Influence of the C93-Y157 pair and substrate-interactions on enzymatic coupling .................................................................................................................... 43 Cyanide binding and EPR spectroscopy of substrate-bound AI- , -, and C93A FeIII-CDO ........................................................................................................ 50 QM/MM computational models of the (CN/Cys)-bound FeIII-CDO active site ............................................................................................................................ 62 Discussion .................................................................................................................... 69 Chapter 3 Oxidative uncoupling in cysteine dioxygenase is gated by a proton-sensitive intermediate ............................................................................................ 73 Introduction ................................................................................................................... 73 Materials and Methods ................................................................................................. 76 Purification ................................................................................................................ 76 Enzyme assays ........................................................................................................ 77 Solvent Isotope Effects ............................................................................................. 78 vii Viscosity studies ....................................................................................................... 78 Data Analysis ............................................................................................................ 79 Results
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