Comparative Structural and Functional Properties of Human and Rat Monoamine Oxidases Público
Wang, Jin (2007)
Published
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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