Chapter 1. Introduction 1.1. Monoamine Oxidase 1.1.1. Characteristics Monoamine oxidase [EC 1.4.3.4. (MAO)] is a flavin adenine dinucleotide (FAD) containing enzyme which catalyzes the oxidation of amines to the corresponding aldehyde and ammonia using O2 as an electron acceptor. The stoichiometry of this oxidation reaction is as shown. The pathway will be discussed later. RCH2NH2 + FAD + O2 + H2O RCHO + FADH2 + HOOH + NH3 MAO is an integral protein found in the outer mitochondrial membrane of both neuronal and nonneuronal tissue, e.g. platelets, placenta, liver, heart and lung.1 One of its primary roles is the regulation of the levels of biogenic amines in the brain and peripheral tissues by catalyzing their oxidative deamination. It occurs in two immunologically and catalytically distinct forms, MAO-A and MAO- B. In some tissues a single form is present. In human, most tissues express both MAO-A and MAO-B, although placenta contains predominantly MAO-A2 and platelets3,4 and lymphocytes4 express only MAO-B. Both forms of MAO seem to be present in most areas of the brain. Monoclonal antibodies specific for each of the forms have shown that MAO-A is located in catecholaminergic cells, including those present in the substantia nigra, while MAO-B is located in serotonergic regions.5,6 MAO-B was also found in astrocytes, a neuronal helper cell population found in all parts of the brain. 1 The two forms are distinguished by their sensitivity to highly selective inhibitors. MAO-A is inhibited by nanomolar concentrations of clorgyline7 (1) while MAO-B is inhibited by nanomolar concentrations of R-(-)-deprenyl8 (2). Cl Cl O H N N H3C 1 2 The notion was once held that MAO-A and MAO-B were the same protein with different lipids attached. However, Pearce and Roth9a have demonstrated that both the A and B forms of human brain MAO are separable with retention of enzymatic activity by simple chromatographic techniques. Further studies where monoclonal antibodies have been raised against each form of the enzyme have provided additional evidence indicating that the two major enzyme sub-types are different proteins.9b The most decisive evidence came from the isolation of cDNA clones that encode MAO-A and MAO-B and determination of their nucleotide sequences.10 The amino acid sequence deduced from these data show a 70% homology. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr in which the obligatory co-factor FAD is covalently linked via a thioether bond between the cysteine residue and the C8- methyl substituent of the flavin moiety (Fig. 1).11 2 Ser-Gly-Gly-Cys-Tyr R N N O N H3C N O Fig. 1. Partial Amino Acid Sequence of MAO Containing the Flavin Moiety The subunit molecular weights of the A and B forms have been calculated to be 59,700 and 58,800 daltons, respectively.12 Although the enzymes have not been crystallized, studies show a ratio of one FAD per 63 kDa (MAO-A) and 57 kDa (MAO-B) human enzyme.13 The above data, in conjunction with the molecular weights obtained from cDNA studies, indicate that MAO consists of either one or more subunits, each of which binds one FAD molecule.14 1.1.2. MAO Substrates Monoamine oxidase catalyzes the oxidative deamination of a variety of biogenic amines7,15,16 including dopamine (3), noradrenaline or norepinephrine (4), adrenaline or epinephrine (5), serotonin (6) and 2- phenylethylamine (7). MAO-A preferentially metabolizes serotonin7 while MAO-B shows a preference for 2-phenylethylamine.16 In addition to the endogenous substrates of MAO, numerous substances of exogenous origin have been shown to be oxidatively metabolized by MAO. Benzylamine17a (8) is a preferred MAO-B substrate while kynuramine17b (9) is a preferential MAO-A substrate. 3 H OH H OH NH2 NH2 NHCH3 HO HO HO OH OH OH 3 4 5 O HO NH2 NH2 N NH2 NH2 NH2 H 6 7 8 9 MAO is thought to catalyze amine oxidation by a single electron transfer pathway (discussed in detail in section 1.4). Abstraction of an a -proton from the intermediate cation radical was suggested to be the rate-limiting step in the oxidation process.25 The absolute stereochemistry of the abstraction of a hydrogen atom from the prochiral methylene group has been determined. Yu et al.18 using dopamine or benzylamine as the substrate have established that both MAO-A and B exhibit the same stereospecificity, that is, the pro-R- hydrogen is abstracted by both forms of MAO. DSi H HO HO D C H C Re NH2 NH2 HO HO 2 2 S-[a - H1]-dopamine R-[a - H1]-dopamine 4 1.1.3. MAO Inhibitors The discovery of the therapeutic activity of monoamine oxidase inhibitors was due to the recognition of an important clinical side effect. Patients who had been treated with a tuberculosis fighting drug, iproniazid, displayed a profound mood elevating side effect. After five years of clinical observations in tuberculosis patients, iproniazid was introduced into clinical medicine as the first antidepressant drug.19 Biochemical studies indicated that iproniazid (10) was a specific and potent inhibitor of MAO.20 The discovery that iproniazid is an inhibitor of MAO activity paved the way to the synthesis and pharmacological assessment of inhibitors of the enzyme. Some of the MAO inhibitors currently in clinical use in certain European countries for treatment of depression include phenelzine (11), tranylcypromine (12), pargyline (13) and isocarboxazid (14). The MAO inhibitors can be divided into three structural classes, namely the acetylenes, the cyclopropylamines and the hydrazines. They exhibit their inhibition properties by a variety of mechanisms including irreversible inhibition and mechanism- based inactivation. A brief review of enzyme inhibition is provided in the next section. NHNH2 CONHNH H NH2 H N 10 11 12 N O N NHNH CH CH 3 3 O 13 14 5 1.2. Enzyme Inhibition Enzyme inhibition is a phenomenon of great practical importance as well as a useful probe for characterizing the molecular features of the active site of the enzyme and other factors that influence enzyme activity. The active site of an enzyme represents a small part of the whole enzyme molecule. Characterization of active centers for enzymes for which no X-ray structure is available is dependent on structure activity relationship (SAR) studies including those utilizing specific enzyme inhibitors which may act reversibly or irreversibly. Before proceeding to a discussion of enzyme inhibition it is important to understand how enzymes function as catalysts in the metabolism of substrate molecules in biochemical reactions and therefore a brief description of enzyme catalysis is included. 1.2.1. Enzyme Catalysts Enzymes are proteins that catalyze biochemical reactions. Enzymes in general tend to be highly selective for the type of reaction they catalyze. The "catalytic site" on the enzyme contains functionalities that are directly involved in the catalysis. The catalytic site is usually a small, localized region of the enzyme surface. The substrate binding site and the catalytic site are usually in close proximity to each other and frequently overlapping. Together they are referred to as the "active site". 1.2.2. Kinetics of Enzyme Catalyzed Reactions As a rule kinetic analysis is done on purified or partially purified enzymes in vitro. Kinetic analysis is a major tool used to investigate the mechanism of enzyme catalysis. One of the main features of kinetic analysis involves the variation in the rate of reaction with substrate concentration. For this purpose a 6 fixed low concentration of the enzyme is used in a series of parallel experiments in which only the substrate concentration is varied. Under these conditions the initial reaction velocity increases until it reaches a maximum value at high substrate concentrations reflecting that all enzyme binding sites are occupied with substrate. Initially it may be assumed that the reaction between enzyme (E) and substrate (S) proceeds through the formation of an enzyme-substrate complex (ES) and that the (ES) is in equilibrium with the free enzyme (E) and substrate (S). The formation of products proceeds only through the (ES) complex. The steady-state kinetic condition may be introduced to eliminate the requirement that the (ES) complex be in equilibrium with the free enzyme and substrate. The steady state evaluation assumes a fixed level of (ES) complex (Scheme 1). The steady state, which is achieved shortly after mixing enzyme and substrate, constitutes the time interval when the rate of reaction is constant with time. The concentration of the (ES) complex is also a constant during this time period. Usually the substrate is present in much higher molar concentrations than the enzyme. Since the initial period of the reaction only is being examined, the free substrate concentration is approximately equal to the total substrate added to the reaction mixture. Scheme 1. Enzyme Catalyzed Reaction of Substrate (S) to Product (P) k1 k2 E + S ES E + P k-1 7 For the purpose of graphical representation of experimental data in an enzyme catalyzed reaction it is convenient to use the Lineweaver-Burke equation derived using the steady state approximation which states: 1/V = (Km/Vmax).(1/[S]) + (1/Vmax). The constant Km is called the Michaelis constant. One of the important relationships is that between Km and the substrate concentration (S). Km can be defined as the concentration of the substrate when the maximum rate of enzyme catalyzed reaction is 50%, i.e. half Vmax. From the Lineweaver-Burke equation, a plot of 1/V (the reciprocal of the initial rate) vs 1/S (the reciprocal of the substrate concentration) should give a straight line.