Mechanism of Oxygen Activation and Hydroxylation By
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MECHANISM OF OXYGEN ACTIVATION AND HYDROXYLATION BY THE AROMATIC AMINO ACID HYDROXYLASES A Dissertation by JORGE ALEXANDER PAVON Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2009 Major Subject: Biochemistry MECHANISM OF OXYGEN ACTIVATION AND HYDROXYLATION BY THE AROMATIC AMINO ACID HYDROXYLASES A Dissertation by JORGE ALEXANDER PAVON Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-Chairs of Committee, Paul F. Fitzpatrick Gary R. Kunkel Committee Members, J. Martin Scholtz Frank M. Raushel Head of Department, Gregory D. Reinhart May 2009 Major Subject: Biochemistry iii ABSTRACT Mechanism of Oxygen Activation and Hydroxylation by the Aromatic Amino Acid Hydroxylases. (May 2009) Jorge Alexander Pavon, B.A., Rutgers University Co-Chairs of Advisory Committee: Dr. Paul F. Fitzpatrick Dr. Gary R. Kunkel The aromatic amino acid hydroxylases phenylalanine hydroxylase (PheH), tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) utilize tetrahydropterin and molecular oxygen to catalyze aromatic hydroxylation. All three enzymes have similar active sites and contain an iron atom facially coordinated by two histidines and a glutamate. The three enzymes also catalyze the benzylic hydroxylation of 4- methylphenylalanine. The intrinsic primary and α-secondary isotope effects for benzylic hydroxylation and their temperature dependences are nearly identical for the three enzymes, suggesting that the transition states, the tunneling contributions and the reactivities of the iron centers are the same. When molecular oxygen and the tetrahydropterin are replaced by hydrogen peroxide (H2O2), these enzymes catalyze the hydroxylation of phenylalanine to form tyrosine and meta-tyrosine with nearly identical second order rate constants. When the H2O2-dependent reaction is carried out with cyclohexylalanine or 4-methylphenylalanine, the products are 4-HO-cyclohexylalanine and 4-hydroxymethylphenylalanine, respectively. These experiments provide further evidence that the intrinsic reactivities of the iron centers in these enzymes are the same. iv Wild-type PheH and the uncoupled mutant protein V379D exhibit normal and inverse isotope effects, respectively, with deuterated phenylalanines. When the reaction is monitored by stopped-flow absorbance spectroscopy, three steps are visible. The first step is the reversible binding of O2, the second step is 5-7 fold faster than the turnover number, setting a limiting value for the rate constant for O2 activation, and the last step is non-enzymatic. There is no burst in the pre-steady state formation of tyrosine. These results are consistent with formation of the new C-O bond to form tyrosine as the rate- limiting step of the reaction. The reaction of TrpH with both tryptophan and phenylalanine was studied by stopped-flow absorbance spectroscopy and rapid-quench product analysis. With either amino acid as substrate, four steps can be distinguished. The first step is the reversible binding of O2 to the Fe(II) center; this results in an absorbance signature with a maximum at 420 nm. This O2 complex decays with a rate constant that is 18-22 fold faster than the turnover number with either amino acid, setting a the lower limit for the rate constant for O2 activation. The rate constant for the third step agrees well with the pre-steady state of formation of 5-hydroxytryptophan or tyrosine from rapid-quench product analysis. The rate constant for the fourth step agrees well with the turnover number. Overall, these results show that O2 activation is fast and turnover with each amino acid is limited by hydroxylation and release of a product, with the former step being about 4-fold faster than the latter. v DEDICATION This dissertation is dedicated to my parents for giving me the gift of life and the opportunity to follow my dreams. vi ACKNOWLEDGEMENTS I would like to thank my advisor and co-chair, Dr. Paul F. Fitzpatrick, for allowing me to be part of his laboratory, for his knowledge, guidance, support and for making my research enjoyable. I would also like to thank all the lab members, past and present; especially Dr. Michael P. Valley, whose work ethic I tried to emulate very early. He told me to read 5-7 research articles every week and more importantly to learn to love your research. I would especially like to thank Michaela Huynh; for the past six months she has been an outstanding researcher partner. She played an important role in chapters III and VI of this dissertation. I would also like to thank Dr. William Park and Dr. Greg Reinhart for everything they have did for me. I would also like to thank past and present committee members: co-chair, Dr. Gary Kunkel, Dr. J. Martin Scholtz, Dr. Frank Raushel and Dr. Michael P. Kladde. I would like to thank Dr. Martin Bollinger and Dr. Carsten Krebs for allowing me to do experiments in their laboratories and discussing subtle details about transient kinetics and Mössbauer spectroscopy with me. Finally, I would like to thank my parents and Dr. Roger Lalancette; without them I would never have gone to graduate school. My parents have always been there for me; they forced me to stay in high school. They taught me to work hard, to be tough on myself, to have goals in life and more importantly to remember that anything worth having in life should never come easy. I would like to thank all my friends at Texas A&M. Last, but certainly not least, my brothers Robert, Davis, David and my sister vii Diana; I learned a lot from all you, especially to always be happy even when everything seems to go wrong. viii NOMENCLATURE TyrH tyrosine hydroxylase PheH phenylalanine hydroxylase TrpH tryptophan hydroxylase Δ117PheH catalytic core of phenylalanine hydroxylase lacking 116 residues from the N-terminus TrpH102-416 catalytic core of tryptophan hydroxylase lacking 101 and 28 residues from the amino and carboxyl termini respectively NDO oxygenase component of napthalene 1,2 dioxygenase BZDOS oxygenase component of benzoate 1,2-dioxygenase DHPR dihydropteridine reductase TauD α-ketoglutarate dependent dioxygenase HPPD (4-hydroxyphenyl) pyruvate dioxygenase Dopa dihydroxyphenylalanine 5-HO-trp 5-hydroxytryptophan 6-MePH4 6-methyl-tetrahydropterin 6Me7,8PH2 6-methyl-7,8-dihydropterin 5Me5DPH4 6-methyl-5-deazatetrahydropterin q6MePH2 quinonoid 6-methyldihydropterin 4a-6MePH3OH 4a-hydroxypterin NDA naphthalene-2,3-dicarboxaldeyde CBI 1-cyanobenz[f]-isoindole ix ESI electrospray negative ion mode mass spectrometry EDTA ethylenediamine tetraacetic acid NTA nitrilotriacetic acid DTT dithiothreitol Hepes N-(2-hydroxyethyl) piperazine-N’-2-ethane-sulfonic acid PKU phenylketonuria x TABLE OF CONTENTS Page ABSTRACT.......................................................................................................... iii DEDICATION ...................................................................................................... v ACKNOWLEDGEMENTS ................................................................................... vi NOMENCLATURE .............................................................................................. viii TABLE OF CONTENTS....................................................................................... x LIST OF FIGURES............................................................................................... xii LIST OF TABLES................................................................................................. xvii CHAPTER I INTRODUCTION............................................................................. 1 II INTRINSIC ISOTOPE EFFECTS ON BENZYLIC HYDROXYLATION BY THE AROMATIC AMINO ACID HYDROXYLASES: EVIDENCE FOR HYDROGEN TUNNELING, COUPLED MOTION AND SIMILAR TRANSITION STATE STRUCTURESMETHODS.......................... 10 Introduction................................................................................. 10 Experimental Procedures ............................................................. 12 Results......................................................................................... 14 Discussion ................................................................................... 22 III DEMONSTRATION OF A PEROXIDE SHUNT FOR THE AROMATIC AMINO ACID HYDROXYLASES............................. 28 Introduction................................................................................. 28 Experimental Procedures ............................................................. 30 Results......................................................................................... 34 Discussion ................................................................................... 47 xi CHAPTER Page IV INSIGHTS INTO THE CATALYTIC MECHANISMS OF PHENYLALANINE AND TRYPTOPHAN HYDROXYLASE FROM KINETIC ISOTOPE EFFECTS ON AROMATIC HYDROXYLATION ........................................................................ 58 Introduction................................................................................. 58 Experimental Procedures ............................................................. 60 Results......................................................................................... 65 Discussion ................................................................................... 70 V INVESTIGATION OF THE CHEMICAL MECHANISM OF PHENYLALANINE HYDROXYLASE BY STOPPED-FLOW ABSORBANCE SPECTROSCOPY,