FLAVIN AMINE OXIDASES FROM THE MONOAMINE OXIDASE STRUCTURAL FAMILY UTILIZE A HYDRIDE TRANSFER MECHANISM A Dissertation by MICHELLE HENDERSON POZZI 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 2010 Major Subject: Biochemistry FLAVIN AMINE OXIDASES FROM THE MONOAMINE OXIDASE STRUCTURAL FAMILY UTILIZE A HYDRIDE TRANSFER MECHANISM A Dissertation by MICHELLE HENDERSON POZZI 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 Gregory D. Reinhart Committee Members, Mary Bryk Frank M. Raushel Head of Department, Gregory D. Reinhart May 2010 Major Subject: Biochemistry iii ABSTRACT Flavin Amine Oxidases from the Monoamine Oxidase Structural Family Utilize a Hydride Transfer Mechanism. (May 2010) Michelle Henderson Pozzi, B.S., Sam Houston State University Co-Chairs of Advisory Committee: Dr. Paul F. Fitzpatrick Dr. Gregory D. Reinhart The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the k cat /K amine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred , displays a pK a of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the k cat /K amine -pH profile with the substrate spermine, and the k red value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in k cat /K O2 . Solvent isotope effects and proton inventories are consistent iv with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para -substituted N,N’-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the k red values for each correlate with the van der Waals volume (V W) and the σ value. The coefficient for V W is the same at pH 8.6 and 6.6, whereas the ρ value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The k cat /K M pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The k cat and k red values are equal, and identical isotope effects are observed on k red, kcat , and k cat /K M, indicating that CH bond cleavage is rate-limiting with this substrate. v DEDICATION This dissertation is dedicated to my family. For my parents and grandmother, who have taught me through their words and actions how to live this life as stated in Philippians 3:14, “I press on toward the goal for the prize of the upward call of God in Christ Jesus.” To my husband for all the support, love, and friendship we share, strengthened by our faith and values to recognize true achievements in life, “Let not a wise man boast of his wisdom, and let not the mighty man boast of his might, let not a rich man boast of his riches; but let him who boasts boast of this, that he understands and knows Me” (Jeremiah 9:23-24). To my son, the highlight of my life, the greatest blessing and responsibility I have ever been given, and the one I want to give all opportunities. And finally to my precious little dogs who faithfully wait for my return from work every day and sit with me during long nights of study. vi ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Paul F. Fitzpatrick for his expertise, guidance and support, and my committee members for their involvement in overseeing my progress throughout my graduate career. I would like to thank all of my lab mates who have been great co-workers and great friends. I would like to thank the many friends I have made in graduate school, who have made the bad times better and the good times great. And finally, I would like to thank my family for their unwavering support, encouragement, love, and most importantly, their prayers. vii ABBREVIATIONS DAAO D-Amino acid oxidase FAD Flavin adenine dinucleotide FMN Flavin mononucleotide Fms1 S. cerevisiae spermine oxidase LAAO L-Amino acid oxidase LSD1 Lysine-specific demethylase 1 MAO Monoamine oxidase MTOX N-Methyltryptophan oxidase PAO Polyamine oxidase SSAT Spermidine/spermine N1-acetyltransferase TMO Tryptophan 2-monooxygenase viii TABLE OF CONTENTS Page ABSTRACT.............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi ABBREVIATIONS................................................................................................... vii TABLE OF CONTENTS .......................................................................................... viii LIST OF FIGURES................................................................................................... x LIST OF TABLES .................................................................................................... xii CHAPTER I INTRODUCTION................................................................................ 1 Polyamine Metabolism and Regulation ......................................... 1 Polyamine Oxidase......................................................................... 6 Flavoprotein Amine Oxidases........................................................ 9 II pH DEPENDENCE OF A MAMMALIAN POLYAMINE OXIDASE: INSIGHTS INTO SUBSTRATE SPECIFICITY AND THE ROLE OF LYSINE 315.............................................................. 19 Experimental Procedures................................................................ 23 Results............................................................................................ 26 Discussion...................................................................................... 39 III LYS315 PLAYS A ROLE IN THE OXIDATIVE-HALF REACTION IN MAMMALIAN POLYAMINE OXIDASE .............. 44 Experimental Procedures................................................................ 47 Results............................................................................................ 49 Discussion ...................................................................................... 54 ix CHAPTER Page IV MECHANISTIC STUDIES OF PARA-SUBSTITUTED N,N’- DIBENZYL-1,4-DIAMINOBUTANES AS SUBSTRATES FOR A MAMMALIAN POLYAMINE OXIDASE......................................... 59 Experimental Procedures................................................................ 62 Results............................................................................................ 66 Discussion...................................................................................... 80 V USE OF pH AND KINETIC ISOTOPE EFFECTS TO ESTABLISH CHEMISTRY AS RATE-LIMITING IN OXIDATION OF A PEPTIDE SUBSTRATE BY LSD1..................................................... 86 Experimental Procedures................................................................ 90 Results............................................................................................ 93 Discussion ...................................................................................... 99 VI SUMMARY ......................................................................................... 103 REFERENCES.......................................................................................................... 107 VITA ......................................................................................................................... 125 x LIST OF FIGURES FIGURE Page 1.1 Polyamine structures .................................................................................. 3 1.2 Polyamine metabolism............................................................................... 5 1.3 PAO oxidation of N1-acetylspermine........................................................ 8 1.4 General reaction mechanism for flavoprotein amine oxidases .................. 10 1.5 Conversion of riboflavin to FMN and FAD............................................... 10 1.6 Electronic conversion of oxidized flavin to semiquinone to reduced flavin............................................................................................. 10 1.7 UV-visible spectra of oxidized flavin (solid line), semiquinone (dotted line), and reduced flavin (dashed line)....................................................... 11 1.8 The SET mechanism .................................................................................. 14 1.9 The polar nucleophilic mechanism