Dopamine 3-hydroxylase

Dopamine /3-hydroxylase is a monoxygenase that catalyzes the hydroxylation of dopamine to form norepinephrine. This enzyme is localized in the chromaffin granules of the adrenal medulla and in the storage vesicles of central and peripheral catecholaminergic neurons. Since these compounds are unstable, this activity is often assayed by following the formation of octopamine from tyramine. For example, in the assay developed by Feilchenfeld et al. (1982), the reactant tyramine was separated from the product octopamine by reversed-phase, ion-paired HPLC (/uBondapak C18 using a mobile phase of 17% (v/v) 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

In contrast, in the assay described by Lee et al. (1987), epinephrine and norepinephrine were separated as the 1,2-diphenylethylenediamine deriva- 

10 fiM copper(II) sulfate, 10 /aL of 10 mg/mL catalase, 10 /aL of 60 m M (for assay of human plasma) or 250 m M (for rat serum) L-ascorbic add, and 10 fiL of 0.3 M sodium fumurate. The reaction was started by the addition of 20 fiL of 80 mM dopamine. After 15 to 30 minutes at 37°C, the reaction was stopped by the addition of 700 /aL of 0.5 M trichloroacetic acid, and 100 fiL of 4 ftM epinephrine was added as an internal standard. After centrifugation, 100 /aL of the supemate was added to a Toyopak 1C-SP M cartridge. The cartridge was washed in succession with 10 mL of water, 2 mL of 0.2 M sodium phosphate buffer (pH 6.0), 2 mL of water, and 2 mL of 50% acetonitrile. The adsorbed amines were eluted with 2 mL of a 1 1 mixture of acetonitrile and 0.6 M KC1. To the eluate were added 200 /aL of 1,2-diphenylethylenediamine solution (212 mg in 0.1 M HQ) and 10 /aL of 0.3 M potassium hexacyanoferrate(III). The mixture was allowed to stand at 37°C for 40 minutes before analysis of 100 /aL by HPLC. The amount of dopamine formed was linear up to 15 /aL of human plasma, and with time up to 30 minutes. 

The assay was validated using rat serum and human plasma from heparinized blood of healthy volunteers. 

---

Methyldopa, through its metaboHte, CX-methyInorepinephrine formed in the brain, acts on the postsynaptic tt2-adrenoceptor in the central nervous system. It reduces the adrenergic outflow to the cardiovascular system, thereby decreasing arterial blood pressure. If the conversion of methyldopa to CX-methyl norepinephrine in the brain is prevented by a dopamine -hydroxylase inhibitor capable of penetrating into the brain, it loses its antihypertensive effects. 

Copper is one of the twenty-seven elements known to be essential to humans (69—72) (see Mineral nutrients). The daily recommended requirement for humans is 2.5—5.0 mg (73). Copper is probably second only to iron as an oxidation catalyst and oxygen carrier in humans (74). It is present in many proteins, such as hemocyanin [9013-32-3] galactose oxidase [9028-79-9] ceruloplasmin [9031 -37-2] dopamine -hydroxylase, monoamine oxidase [9001-66-5] superoxide dismutase [9054-89-17, and phenolase (75,76). Copper aids in photosynthesis and other oxidative processes in plants. 

Dopamine- -hydroxylase (from bovine adrenal medulla) [9013-38-1] Mr -290,000, [EC... 

Dopamine hydroxylase synthesizes norepinephrine (Nor-adrenalin) from dopamine. 

Neurotransmitter Transporters. Figure 3 Dopamine turnover at a presynaptic nerve terminal, (a) Dopamine is produced by tyrosine hydroxylase (TH). When secretory vesicles are filled, they join the releasable pool of vesicles at the presynaptic membrane. Upon exocytosis, the diffusion of released dopamine is limited by reuptake via DAT. (b) If DAT is inactive, dopamine spreads in the cerebrospinal fluid but cannot accumulate in secretory vesicles. This results in a compensatory increase of dopamine hydroxylase activity and a higher extracellular dopamine level mice with inactive DAT are hyperactive. 

C. Dopamine -Hydroxylase (DBH) Catalyzes THE Conversion of Dopamine to Norepinephrine... 

DBH, dopamine / -hydroxylase DRD2, dopamine D2 receptor DRD4, dopamine D4 receptor CYP2A6, cytochrome P450 2A6 CYP2D6, cytochrome P450 2D6. 

Cysteine string protein (CSP) Cytochrome b561 Peripheral membrane protein that is paimitoylated on >10 cysteines. May have a role in Ca2+ sensitivity of exocytosis. Electron-transport protein required for intravesicular monooxygenases in subsets of secretory vesicles. Required for dopamine- -hydroxylase and peptide amidase activity. 

Methyldopa is an false substrate for the dopamine-/ -hydroxylase resulting in a-methylnor-adrenaline. This metabolite is an a2-adrenoceptor agonist an induce, like clonidine, a centrally mediated reduction of sympathetic tonus. 

E. Lui, R. Chirakal, G. Firnau, Enzymatic synthesis of (-)-6-[ F]-fluoronorepinephrine from 6-[ F]-fluorodopamine by dopamine -hydroxylase, J. Label. Compd. Radio-pharm. 41 (1998) 503-521. 

Dunnette J, Weinshilbourn RM (1977) Inheritance of low immunoreactive human plasma dopamine hydroxylase. Radioimmunoassay studies. J Clin Invest 609 1080... 

DMF, in alkylation of aminothiazoles, 35 in ambident reactivity, 36 in cyclizations, 50 DMSO, reaction with CS2,61 Dopamine content in brain, 146 Dopamine hydroxylase inhibitor, 152 Double bond, addition to, 40 ozonolysis of, 71... 

The secretion of epinephrine by the adrenal medulla is controlled directly by nerve impulses and also by the other stress hormones, namely, corticosteroids. This is illustrated in Figure 16.11. Nerve impulses have a major stimulatory effect on tyrosine and dopamine hydroxylases, whereas glucocorticoids have a major effect on phenylethanolamine methyltransferase. Tyrosine hydroxylase is considered the rate-controlling enzyme in the biosynthesis... 

Figure 16.11 Control of catecholamine biosynthesis in the adrenal medulla. TH, tyrosine hydroxylase DBH, dopamine hydroxylase PNMT, phenylethanolamine methyl-transferase ACTH, adrenocorticotropic hormone. The heavy arrows indicate major sites of regulation. (Reproduced by permission from Axelrod, J. Reisine TD. Stress hormones their interaction and regulation. Science 224 452-459, 1984.)...

Dopamine hydroxylase belongs to the copper-dependent monooxygenases and is important in the control of the neurotransmitter concentrations of dopamine and norepinephrine [59]. 

Figure 13.4. Synthesis of the catecholamines. Tyrosine hydroxylase, EC 1.14.16.2 (see also Fignre 10.10) aromatic amino acid decarboxylase, EC 4.1.1.26 and dopamine -hydroxylase, EC 1.14.17.1.

Dopamine -hydroxylase is a copper-containing enzyme involved in the synthesis of the catecholamines noradrenaline and adrenaline from tyrosine in the adrenal medulla and central nervous system (see Figure 13.4). The active enzyme contains Cu+, which is oxidized to Cu + during the hydroxylation of the substrate. Reduction back to Cu+ specifically requires ascorbate, which is oxidized to monodehydroascorbate. 

The term scurvy is derived from the Italian scorbutica, meaning an irritable, neurotic, discontented, whining, and cranky person. The deficiency disease is certainly associated with listlessness and general malaise, and sometimes changes in personality and psychomotor performance and a lowering of the general level of arousal. The behavioral effects can presumably be attributed to impaired synthesis of catecholamines as a result of reduced activity of dopamine -hydroxylase (Section 13.3.1). 

Ascorbic acid has specific and well-defined roles in two classes of enzymes the copper-containing hydroxylases (such as dopamine -hydroxylase and peptidyl glycine hydroxylase) and the 2-oxoglutarate-Unked iron-containing hydroxylases, of which the best studied are the proUne and lysine hydroxylases involved in maturation of connective tissue (and other) proteins. 

The clinical symptoms of classical Menkes disease can be traced back to developmentaUy important copper enzymes such as lysyl oxidase, tyrosinase (see Copper Hemocyanin/Tyrosinase Models), cytochrome c oxidase (see Cytochrome Oxidase), dopamine -hydroxylase, superoxide dismutase, and amine oxidase (see Superoxide Dismutase). Lysyl oxidase is needed for the cross-linking of connective tissue a deficiency in this enzyme causes weakened connective tissue and connective tissue disorder such as arterial ruptures as observed in these patients. Low levels of cytochrome c oxidase cause temperature instability and the absence of tyrosinase explains the hair depigmentation observed in affected individuals. ... 

A number of copper requiring enzymes are located at the cell surface or are exported into the extracellular milieu. Examples of such secretory Cu-enzymes include copper requiring ferroxidases that fimction in iron transport (e g. ceruloplasmin, CP), enzymes for neurotransmission (peptidyl amidating enzyme and dopamine hydroxylase), an extracellular superoxide dismutase (SOD) that fimctions in antioxidant defense and enzymes for formation of connective tissue (lysyl oxidase), and pigments (tyrosinase) (reviewed in ). En route to their designated location, each of these enzymes passes through a specialized compartment of the late Golgi where copper insertion takes place. 

Tyrosine monooxygenase uses biopterin as a cofactor. Biopterin is made in the body and is not a vitamin. Its structure resembles that of folic acid. Dopa decarboxylase is a vitamin B -requiring enzyme. Dopamine hydroxylase is a copper metalloenzyme. The active form of the enzyme contains copper in the reduced state (cuprous, Cu+). With each catalytic event, the copper is oxidized to the cupric state (Cu ). The enzyme uses ascorbic acid as a cofactor for converting the cupric copper back to cuprous copper. Thus, each catalytic event also results in the conversion of ascorbic acid to semidehydroascorbate. The semidehydroascorbate, perhaps by disproportionation, is converted to ascorbate and dehydroascorbate. The catalytic cycle of dopamine hydroxylase is shown in Figure 9,86. Dopamine hydroxylase, as well as the stored catecholamines, are located in special vesicles... 

FIGURE 9.86 Ascorbate is requited for the activity of dopamine hydroxylase, also called dopamine-p-monooxygenase. The mechanism of the reaction is quite similar to that of amidating en2)nne, an ascorbate-requiring enzyme that catalyzes the hydroxylation of pol)q)eptides, during the course of a two-step sequence. 

---