Comparative Structural and Functional Properties of Human and Rat Monoamine Oxidases Público

Abstract

Monoamine oxidases A and B (MAO A and MAO B) are flavin-containing mitochondrial membrane-bound proteins which function in the oxidative degradation of biogenic and xenobiotic amines to their corresponding aldehydes. The crystal structures of MAOs show that human MAO A is monomeric, whereas human MAO B and rat MAO A are dimeric. Recent sequencing and modeling studies suggest a rationale for these differences in that human MAO A exhibits a selective mutation in the region close to the membrane surface and the dimer interface where Glu151 is a Lys in human MAO A but remains a Glu in human MAO B and non-human MAO A's. This dissertation describes the experiments to probe the structures of purified human MAO A and MAO B which are compared with the crystal structures solved by X-ray crystallography, as well as to investigate functional consequences of the Glu151Lys selective mutation in human MAO A. Human MAO A and MAO B were labeled with a fluorophore donor attached to the acetylenic inhibitor pargyline in the active site cavities and with thiol-reactive acceptor reagent on a cysteine either on the surface of the protein or at the end of the C-terminal helices. FRET analysis shows that the structures of both human MAO A and MAO B in detergent solution are consistent with their respective crystal structures. Furthermore, the C-terminal helices of these two enzymes are suggested to exhibit a non-linear conformation in which the ends turn back toward the membrane surface. Rat MAO A was successfully expressed in P. pastoris system and purified according to the modified human MAO A purification procedure. Even though these two enzymes are 90% sequence identical, different surface charges on the proteins result in different binding affinities on ion-exchange columns. An electrostatic potential study on human and rat MAO A's shows that the distribution of negatively charged residues is greater on human MAO A than on rat MAO A, resulting in a stronger binding of human MAO A to the anion-exchange DEAE-Sepharose column. Comparisons of human wild-type MAO A, human MAO A Lys151Glu mutant and rat MAO A show that there are similar functional properties among them except thermal stability. Both human MAO A Lys151Glu mutant and rat MAO A exhibit a much higher thermal stability than does human wild-type MAO A.

Table of Contents

 

Chapter 1 Introduction....................................................................................................... 1

1.1 General introduction to monoamine oxidases.................................................................... 1

1.1.1 Reactions catalyzed by MAOs and their isoforms................................................... 1

1.1.2 Tissue and cell distribution..................................................................................... 2

1.2 Pharmacological significance............................................................................................ 3

1.2.1 Substrate and inhibitor similarities and specificities.................................................. 3

1.2.2 Physiological role in health and disease................................................................... 6

1.2.2.1 MAO A and B in psychiatric disorders................................................... 7

1.2.2.2 MAO A and B in Parkinson's disease..................................................... 8

1.2.2.3 MAO A and B in smoking and alcoholism............................................... 9

1.3 Molecular genetics and biochemistry.............................................................................. 10

1.3.1 Gene cloning and location.................................................................................... 10

1.3.2 Protein expression system.................................................................................... 13

1.3.3 Covalent FAD cofactor....................................................................................... 14

1.3.4 Catalytic mechanism............................................................................................ 18

1.3.4.1 Aminium cation radical mechanism........................................................ 19

1.3.4.2 Polar nucleophilic mechanism................................................................ 20

1.3.4.3 Concerted nucleophilic mechanism........................................................ 22

1.3.5 Quantitative structure-activity relationships (QSAR)............................................. 23

1.4 Structural biology........................................................................................................... 25

1.4.1 Human MAO B.................................................................................................. 26

1.4.2 Rat MAO A........................................................................................................ 32

1.4.3 Human MAO A.................................................................................................. 34

1.4.4 Selective mutation in human MAO A................................................................... 38

1.5 Characterization of mitochondrial membrane associated monoamine oxidase................... 40

1.5.1 Lipid composition of mitochondria....................................................................... 40

1.5.2 Effect of phospholipids on functional properties of MAO...................................... 42

1.5.3 Targeting signal and membrane association of MAO............................................. 43

1.6 Dissertation objectives................................................................................................... 44

 

Chapter 2 Fluorescent Probes to Investigate the Structural Properties of Human MAO A and MAO B      47

2.1 Introduction................................................................................................................... 47

2.2 Materials and Methods.................................................................................................. 50

2.2.1 Materials............................................................................................................. 50

2.2.2 Creation and transformation of site-specific double mutants of MAO A and MAO B 51

2.2.3 Expression and purification of human MAO A and human MAO B double mutants 52

2.2.4 Synthesis of 5'-(N-dansyl) -cadaveryl-p-carboxymethylpargyline (DCP).............. 52

2.2.5 Time course of wild-type MAO inhibition by DCP............................................... 52

2.2.6 Labeling of human MAO enzymes with DCP....................................................... 54

2.2.7 Labeling of DCP-labeled human wild-type MAO A/B and double mutants with 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI)................................... 54

2.2.8 Fluorescence measurements................................................................................. 55

2.2.9 Fluorescence resonance energy transfer (FRET) analysis...................................... 57

2.2.10 Modeling of MAO C-terminal helices................................................................ 58

2.2.11 Molecular Dynamics (MD) simulation................................................................ 59

2.3 Results.......................................................................................................................... 60

2.3.1 Spectral characterization of DCP......................................................................... 60

2.3.2 Inhibition of MAO with DCP............................................................................... 61

2.3.3 Fluorescence quenching of DCP in MAO............................................................ 64

2.3.4 Polarization and anisotropy of DCP in MAO........................................................ 67

2.3.5 Distance measurements in MAO.......................................................................... 68

2.4 Discussion..................................................................................................................... 74

 

Chapter 3 Conformational Investigations of C-terminal Helices of Human MAO A and MAO B in Their Membrane Bound Forms............................................................................... 78

3.1 Introduction................................................................................................................... 78

3.2 Materials and Methods.................................................................................................. 82

3.2.1 Materials............................................................................................................. 82

3.2.2 Creation of human MAO A Cys266Ala/Pro525Cys and MAO B Cys5Ala/Val518Cys mutants 82

3.2.3 Labeling of human MAO A and MAO B double mutants with N-(1-pyrenyl)maleimide (NPM)  82

3.2.4 Isolation of yeast mitochondria............................................................................. 83

3.2.5 Reconstitution of wild-type MAO and NPM-labeled MAO samples into yeast mitochondria       84

3.2.6 Fluorescence measurements................................................................................. 85

3.3 Results.......................................................................................................................... 86

3.3.1 Characterization of MAO insertion into yeast mitochondria................................... 86

3.3.2 Fluorescence spectra of pyrene in MAO.............................................................. 91

3.3.3 Polarization and anisotropy of NPM-labeled MAO in detergent solubilized and membrane bound forms    93

3.3.4 Quenching of pyrene-labeled MAO A and MAO B fluorescence by iodide.......... 94

3.3.5 Quenching of pyrene-labeled MAO A and MAO B fluorescence by spin-labeled fatty acids       97

3.4 Discussion................................................................................................................... 101

 

Chapter 4 High-level Expression of Rat Monoamine Oxidase A in Pichia pastoris........

......................................................................................................................................... 107

4.1 Introduction................................................................................................................. 107

4.2 Materials and Methods................................................................................................ 108

4.2.1 Materials........................................................................................................... 108

4.2.2 cDNA cloning of rat MAO A............................................................................ 108

4.2.3 Transformation of rat MAO A gene into P. pastoris.......................................... 109

4.2.4 Expression of rat MAO A................................................................................. 109

4.2.5 Purification of rat MAO A................................................................................. 109

4.2.6 Mass spectral analysis....................................................................................... 111

4.2.7 Thermal stability................................................................................................ 112

4.2.8 Steady-state kinetic studies................................................................................ 112

4.3 Results........................................................................................................................ 113

4.3.1 Enzyme expression and purification.................................................................... 113

4.3.2 Protein characterization...................................................................................... 114

4.3.3 MALDI-TOF/TOF-MS analysis....................................................................... 114

4.3.4 Functional characterization................................................................................. 116

4.3.5 Thermal stability................................................................................................ 118

4.3.6 Catalytic properties........................................................................................... 120

4.4 Discussion................................................................................................................... 120

 

Chapter 5 Functional Comparison of Human MAO A K151E and Rat MAO A with WT-human MAO A     124

5.1 Introduction................................................................................................................. 124

5.2 Materials and Methods................................................................................................ 125

5.2.1 Materials........................................................................................................... 125

5.2.2 Creation of and transformation of human MAO A K151E.................................. 125

5.2.3 Expression and purification of human MAO A K151E....................................... 126

5.2.4 Thermal stability................................................................................................ 126

5.2.5 Determination of kcat, Km and Ki........................................................................ 127

5.2.6 Determination of Km(O2)................................................................................... 127

5.2.7 Steady state kinetic measurements of para-substituted benzylamine analogue oxidation 127

5.2.8 Data analysis..................................................................................................... 128

5.3 Results........................................................................................................................ 128

5.3.1 Enzyme expression and purification.................................................................... 128

5.3.2 Spectral property of human MAO A K151E..................................................... 129

5.3.3 Thermal stability................................................................................................ 129

5.3.4 Steady state kinetic properties and competitive inhibition.................................... 132

5.3.5 Km(O2)............................................................................................................. 132

5.3.6 Steady state kinetics of the human MAO A K151E and rat MAO A catalyzed oxidation of para-substituted benzylamine analogues and the effects of isotopic substitution............................. 137

5.3.7 Effect of the para substituent on the rates of steady state turnover....................... 139

5.3.8 Quantitative structure-activity relationships describing the binding of para-substituted benzylamine analogues to MAO A's......................................................................................................... 151

5.4  Discussion................................................................................................................... 161

5.4.1 Thermal stability and substrate/inhibitor specificities............................................ 161

5.4.2 Mechanistic interpretation of Km(O2)................................................................. 161

5.4.3 Structure-activity relationships describing the steady-state turnover rate of human MAO A K151E mutant and rat MAO A by para-substituted benzylamine analogues.......................................... 162

5.4.4 Structure-activity relationships describing the binding of para-substituted benzylamine analogues to human MAO A K151E mutant and rat MAO A..................................................................... 164

5.4.5 Mechanistic interpretation of QSAR results........................................................ 165

Dissertation summary.................................................................................................... 171

References...................................................................................................................... 176

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Palabra Clave	human rat monoamine oxidase
Committee Chair / Thesis Advisor	Edmondson, Dale E, Emory University
Committee Members	Lutz, Stefan, Emory University Justice Jr., Joseph B, Emory University

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