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The Pennsylvania State University the Graduate School Eberly College of Science The Pennsylvania State University The Graduate School Eberly College of Science MECHANISTIC DISSECTION OF TAURINE α-KETOGLUTARATE DIOXYGENASE (TauD): A MODEL α-KETOGLUTARATE DIOXYGENASE A Thesis in Biochemistry, Microbiology, and Molecular Biology by John C. Price © 2005 John C. Price Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2005 Thesis of John C. Price was reviewed and approved∗ by the following: Joseph M. Bollinger, Jr. Associate Professor of Biochemistry and Molecular Biology and Associate Professor of Chemistry Thesis Co-advisor Co-chair of Committee Carsten Krebs Assistant Professor of Biochemistry and Molecular Biology and Assistant Professor of Chemistry Thesis Co-advisor Co-chair of Committee Squire J. Booker Associate Professor of Biochemistry and Molecular Biology and Associate Professor of Chemistry Craig E. Cameron Paul Berg Professor of Biochemistry and Molecular Biology Michael T. Green Assistant Professor of Chemistry Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology ∗ Signatures are on file in the Graduate School iii Abstract The oxidizing power of O2 is the basis for the function of the respiratory cycle and is used in many biosynthetic processes. Harnessing these oxidizing equivalents requires levels of precaution and precise control, even so preventing and repairing the damage due to reduced oxygen species is a constant effort in aerobic organisms. The Fe(II)•α-ketoglutarate dioxygenase enzymes harness the oxidizing equivalents of O2 in a mechanism which requires the decarboxylation of α-ketoglutarate to create a highly oxidized Fe center. These enzymes are very effective at specific two electron oxidations of unactivated carbon atoms, and occupy key positions in a surprisingly large number of biological systems. This study describes the mechanistic dissection of a model Fe(II)•α-ketoglutarate dioxygenase, taurine dioxygenase (TauD). Contributions made in the study of this system have experimentally expanded our understanding of these enzymes and verified theories postulated more than 20 years ago. Chapter 1 introduces general features of the enzyme family, and summarizes the literature describing what was known prior to this study regarding the mechanism of action for these enzymes. Chapter 1 also introduces several members of the family, using these systems to describe the utility of the family and the importance of understanding their mechanism of action. Chapter 2 presents all the work done in kinetically characterizing the model system (TauD) describing formation of the reactive enzyme complex and detection of intermediates that accumulate within the catalytic cycle. Chapter 3 delineates the use of sophisticated spectroscopy and a synthetic substrate isotopomer to chemically and structurally characterize the two accumulating intermediates. Chapter 4 explores the effect of substrate binding and the unproductive reaction(s) that occur in the absence of substrate. Chapter 5 contains studies from a variety of alternative substrates which offer interesting, yet not fully developed insights into the delicate balance that allows the active site to activate oxygen and successfully utilize the resulting oxidizing equivalents so effectively and specifically. iv Table of Contents List of Figures…………………………………………………………………………...viii List of Tables………………………………………………………………………...…xvii Acknowledgements………………………………………………………………….xviii Chapter 1 Introduction………………………….………………………………...1 General features of the Fe(II)•αKG dioxygenase family…………….….1 HIFα hydroxylases……………………………………………………….2 AlkB……………………………………………………………………...3 Clavaminate synthase……………………………………………………4 SyrB2…………………………………………………………………….4 Prolyl-4-hydroxylase.................................................................................5 Consensus mechanism for the Fe(II)•αKG dioxygenase family………..6 TauD……………………………………………………………………..9 References……………………………………………………………….24 Chapter 2 Kinetic dissection of the catalytic mechanism of taurine•α-ketoglutarate dioxygenase……………………………..………………………………..31 Abstract………………………………….……………………………….32 Introduction……………………………….……………………………...33 Materials and Methods…………………………………………………...34 Construction of TauD overexpression system……………..………...35 Overexpression of TauD………………………...…………...………35 Purification of TauD…………………………………………..……..36 Fe(II) titration…………………………………………………….….37 Determination of steady state sulfite production rate.…………….....37 Determination of steady state CO2 production rate……………….....38 Kinetics of CO2 in a single turnover…………………………………39 Mössbauer spectroscopy and data analysis………………………..…40 v Stopped-flow experiments and simulations……………………….....40 Preparation of Mössbauer samples………………………………..…41 Results…………………………………………………………………....42 Determination of optimum Fe/TauD ratio and substrate concentrations......................................................................................42 Changes at the active site upon binding of substrate (Mössbauer)…..42 Kinetics of substrate binding………………………………………...43 Stopped-flow evidence for two intermediates…………………….....45 Trapping J and its’ characterization by Mössbauer...…….………….45 Simulation of stopped-flow and steady state rate……………………46 Nature of M…………………………………………………………..47 Kinetics of CO2 evolution in a single turnover………………………49 [O2] dependence on the formation of J………………………………50 Substrate binding rates……………………………………………….52 Explanation of slow reformation for quaternary complex….………..53 Discussion………………………………………………………………..54 Formation of reactive TauD complex………………………………..54 Kinetic description of the reaction with O2…………………………..55 Identity of novel Fe intermediate J…………………………………..58 References………………………………………………………………..83 Chapter 3: Characterization of two accumulating intermediates in the catalytic cycle of taurine•α-ketoglutarate dioxygenase…………………………………….88 Abstract…………………………………………………………………..91 Introduction………………………………………………………………92 Materials and methods…………………………………………………..93 2 Synthesis of 1,1-[ H]2-taurine (D-taurine)………………………...…93 vi High-field Mössbauer spectroscopy and analysis…………………..96 EPR spectroscopy……………………………………………………96 Cryoreduction by low temperature γ-radiolysis……………………..96 Results……………………………………………………………………96 Transient state CO2 evolution from D-taurine containing complex…96 Identification of J as hydroxylating intermediate……………………97 2- Uncoupling of CO2 and SO3 production……………………………98 Freeze-quench Mössbauer…………………………………………...99 High-field Mössbauer………………………………………………100 Cryoreduction of J to Fe(III)……………………………………….100 Structural characterization of J……………………………………..102 Nature of rate limiting step…………………………………………103 Discussion………………………………………………………………104 Reference……………………………………………………………….122 Chapter 4: Probing the mechanism of “untriggered” O2 activation by the TauD•Fe(II)•αKG ternary complex……………………………………126 Abstract…………………………………………………………………126 Introduction……………………………………………………………..127 Materials and methods………………………………………………….129 Results…………………………………………………………………..129 Stopped flow evidence for intermediates…………………………...130 Trapping Fe intermediate and characterization by Mössbauer…..…131 Discussion………………………………………………………………131 References………………………………………………………………141 Chapter 5: Use of substrate analogues in the dissection of the taurine•α-ketoglutarate dioxygenase reaction……………………………………………………143 vii Abstract…………………………………………………………………143 Introduction……………………………………………………………..143 Materials and Methods………………………………………………….145 Synthesis of 1,1-[F]2-taurine (F-taurine)……………………………145 Synthesis of N-oxalylglycine……………………………………….148 Synthesis of 4-Oxo-4-thiocarboxy butyric acid…………………….148 Formation of TauD•Fe(III)•αKG•taurine•NO complex……………150 Use of Peracetic acid as an O atom donor………………………….151 Double mixing stopped-flow absorbance experiments……………..151 Results and Discussion…………………………………………………151 1,1-[F]2-taurine (F-taurine) in TauD quaternary complex………….151 4-oxo-4-thiocarboxy-butyric acid as an analog of αKG……………157 N-oxalylglycine as an analog of the αKG………………………….161 Peracetic acid as an O-atom donor to form J6……………………...163 NO as an O2 analog…………………………………………………165 References………………………………………………………………202 Appendix: Spectroscopic characterization of synthetic routes………………………....208 viii List of Figures Scheme 1-1: Generalized reaction of the Fe(II)•αKG dioxygenase family of enzymes……………………………………………………………………………14 Scheme 1-2: Generalized concensus mechanism of the Fe(II)•αKG dioxygenase family……………………………………………………………………………15 Scheme 2-1: The current working hypothesis for the TauD chemical mechanism……65 Scheme 2-2: Kinetic mechanism used to simulate the kinetics of the first intermediate determined……………………………………………………………………………66 Scheme 2-3: Kinetic mechanisms used to simulate the data in Figure 2-10……………67 Scheme 3-1: Mapping of the minimal kinetic mechanism for TauD onto the chemical mechanism………………………………………………………………………….107 Scheme 3-2: Kinetic mechanism used to simulate the kinetics of the first intermediate incorporating the kinetic isotope effect……………………………………………108 2 Scheme 3-3: Synthetic route to 1,1-[ H]2-taurine (D-taurine)………………………..109 14 2- Scheme 3-4: Uncoupling of CO2 evolution from SO3 production………………...110 Scheme 5-1: Synthesis of kinetic mechanism with the chemical mechanism………..168 Scheme 5-2: Synthetic route to 1,1-[F]2-taurine (F-taurine)………………………….169 Scheme 5-3: Synthetic route to N-oxalylglycine (NOG)……………………………..170 Scheme 5-4: Synthetic route to 4-Oxo-4-thiocarboxy-butyric acid (S-αKG)………….171
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