Enzyme dopamine -monooxygenase

Analogous side-chain oxidations occur in various biosynthetic pathways. The neurotransmitter norepinephrine, for instance, is biosynthesized from dopamine by a benzylic hydroxylation reaction. The process is catalyzed by the copper-containing enzyme dopamine -monooxygenase and occurs by a radical mechanism. A copper-oxygen species in the enzyme first abstracts the pro-R benzylic hydrogen to give a radical, and a hydroxyl is then transferred from copper to carbon. 

Figure 11.17 Supplementation of diet with y-linolenic acid to overcome a deficiency of A desaturase Supplementation of a diet with DOPA to overcome a deficiency of monooxygenase in Parkinson s disease. A desaturase is a rate-limiting enzyme in the synthesis of arachidonic acid. Supplementation of diet with y-linolenic acid bypasses this enzyme. Damage to neurones in the brain that use dopamine as a neurotransmitter causes a deficiency of rate-limiting a supplement - enzyme, tyrosine monooxygenase, which is bypassed by a supplement, DOPA (dihydroxyphenylalanine). DOPA (usually, described as L-DOPA) is considered by the medical profession as a drug but, in reality, it is a dietary supplement.

In the experiments with galactose oxidase and dopamine -monooxygenase very low enzyme concentrations have often been used. The addition of an inert protein might help to approximate the natural conditions by providing a protection against surface denaturation and against radicals, and by regulating the trace metal concentrations. 

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... 

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

Central Nervous System. Dopamine monooxygenase (DMO) is an enzyme that requires copper, as a cofactor and uses ascorbate as an electron donor. This enzyme catalyzes the conversion of dopamine to norepinephrine, the important neurotransmitter. There are soluble and membrane-bound forms of the enzyme, the latter being found in the chromaffin granules of the adrenal cortex. Monoamine oxidase, one of the numerous amine oxidases, is a copper-containing enzyme that catalyzes the degradation of serotonin in the brain and is also involved in the metabolism of the catecholamines. 

A third category of copper enzymes are monooxygenases which, in addition to reducing O2 to H2O, add oxygen to the substrate. For example dopamine-j8-hydrox-ylase hydroxylates the benzylic position of dopamine (LIV) to give noradrenalin, one... 

Dopamine / -monooxygenase (D/3M, EC 1.14.17.1) is an enzyme found in mammalian brains that catalyzes the aerobic hydroxylation of dopamine to norepinephrine (Equation (3))." Ascorbate is the physiological electron donor to the enzyme, so that two equivalents of ascorbate are... 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

The first step is catalysed by the tetrahydrobiopterin-dependent enzyme tyrosine hydroxylase (tyrosine 3-monooxygenase), which is regulated by end-product feedback is the rate controlling step in this pathway. A second hydroxylation reaction, that of dopamine to noradrenaline (norepinephrine) (dopamine [3 oxygenase) requires ascorbate (vitamin C). The final reaction is the conversion of noradrenaline (norepinephrine) to adrenaline (epinephrine). This is a methylation step catalysed by phenylethanolamine-jV-methyl transferase (PNMT) in which S-adenosylmethionine (SAM) acts as the methyl group donor. Contrast this with catechol-O-methyl transferase (COMT) which takes part in catecholamine degradation (Section 4.6). 

Such a species cannot be ruled out in reactions of iron-EDTA complexes with hydroperoxides recently described by Bruice and coworkers (27). On the other hand, a hydroperoxide complex that reacts with the substrate such that bond formation fiom O to substrate is concerted with 0-0 bond breaking, as proposed by Klinman for dopamine P-monooxygenase (18), could provide compensation for the cost of 0-0 bond cleavage in the transition state. In fact, it is interesting to speculate that for each of these enzymes, the mechanism by which the substrate is oxidized may be dependent on the reactivity of the substrate. One could envision certain substrates that would react with the metal-bound hydroperoxide ligand prior to or concerted with 0-0 bond cleavage. This possibility is difficult to assess because of our lack of information concerning the reactivity of HQ2" when complexed to different metal ions. 

It has been suggested that the activation of dopamine 3-monooxygenase by ascorbate was due to the formation of OJ by reduction of O, as the enzyme was activated by O J and inhibited by BESOD This inhibition of the enzyme could not be confirmed Dopamine P-monooxygenase could, moreover, be activated... 

The autoxidation of ascorbate, a cosubstrate of dopamine P-monooxygenase, induces the degradation of most proteins including catalase and dopamine p-monooxygenase, but with the exception of (Cu,Zn)-SOD. Catalase protects dopamine P-monooxy-genase and is therefore generally added in the assay systems . The apparent activation or rather the stabilization of the enzyme (6.5 pg) by small amounts of catalase (3.1 pg) was enhanced by native but not by boiled SOD (100 pg) and also by similar amounts of serumalbumin (100 pg) or of boiled catalase (65 pg)... 

The effect of SOD points to the intervention of Oj in the autoxidation of ascorbate. Proteins in large amounts could react with the strong oxidizing agent formed in this Udenfriend s system , thus protecting the enzyme. The ineffectiveness of boiled SOD could be due to its amino-acid composition (See Sect. 4.1.2). While O did not inactivate dopamine P-monooxygenase the rate pf inactivation in the presence of... 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase function. One of the best understood of these is the peptidylglycine a-hydroxylating monoxygenase, which catalyzes the first step of the reaction of Eq. 10-11. The enzyme is a colorless two-copper protein but the copper atoms are 1.1 nm apart and do not form a binuclear center.570 Ascorbate is an essential cosubstrate, with two molecules being oxidized to the semidehydro-ascorbate radical as both coppers are reduced to Cu(I). A ternary complex of reduced enzyme, peptide, and 02 is formed and reacts to give the hydroxylated product.570 A related two-copper enzyme is dopamine (J-monooxygenase, which utilizes 02 and ascorbate to hydroxylate dopamine to noradrenaline (Chapter 25).571/572 These and other types of hydroxylases are compared in Chapter 18. 

In vivo tolerance to copper is quite high, however, deficiency and excess are serious problems. Infants are particularly vulnerable as they take time to assimilate the correct levels and it is known that trace copper from cooking utensils or water pipes can cause childhood cirrhosis. Copper deficiency leads to arterial weakness and heart enlargement. This is probably caused by a decrease in catecholamine neurotransmitters derived from the biosynthesis of adrenaline which requires the copper-containing enzymes phenylalanine hydroxylase, dopamine P-monooxygenase and tyrosinase. 

Klinman JP. The copper-enzyme family of dopamine 3-mono-oxygenase and peptidylglycine a-hydroxylating monooxygenase resolving the chemical pathway for substrate hydroxylation. J. Biol. Chem. 2006 281 3013-3016. 

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. 

FIGURE 9.87 Ascorbate is used for the synthesis of amidated hormones. Two molecules of ascorbate are used for the production of one molecule of amidated hormone, where each ascorbate is converted to semidehydroascorbic acid. Glyoxylate is a b)rproduct of the reaction. The reaction occurs in two steps, which are catalyzed by a bifunctional enzyme, as revealed in the text. The chemistry of the first reaction, which results in the hydroxyla-tion of the substrate, is quite similar to that catalyzed by dopamine-p-monooxygenase. 

The second strategy is utilised by two other enzymes with a type 2 Cu centre, which in addition require ascorbate. Peptidyl-glycine a-hydroxylating monooxygenase (PMH), which converts C-terminal glycine-extended peptides to their a-hydroxylated products (Equation 1), and dopamine p-hydroxylase (OPH), which converts dopamine to noradrenahne (Equation 2) ... 

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