Dihydroxyphenylpyruvic acid

The intermediate formation of 2,5-dihydroxyphenylpyruvic acid in this conversion has not been proved by isolation. But as this is readily metabolized by tyrosine-oxidizing systems (e.g., 489), unlike the possible alternative intermediate p-hydroxyphenylacetic acid, the pathway is not in doubt. On the other hand, the detailed mechanism of this conversion is probably the major unsolved problem in the study of tyrosine metabolism. 

Uchida, Suzuki, and Ichihara (878) isolated a soluble enzyme system (thereby possibly excluding mitochondrial participation) from rabbit liver, and partially purified it. Two enzymes were involved. The first of these converted p-hydroxyphenylpyruvic acid to 2,5-dihydroxyphenylpyruvic acid. If this enzyme was resolved, vitamin C alone did not restore the activity, but vitamin C and vitamin B12 did. The amount of B12 required was very low, and they suggested that the true enzyme was a Bw derivative, possibly aquocobalamin hydroxide bound to enzyme protein, and that the function of the ascorbic acid was solely to stabilize the reactive form of the coenzyme. This agrees with the work of La Du and Greenberg (524), who considered the role of ascorbic acid to be quite unspecific. Ascorbate increased the rate of tyrosine oxidation in liver preparations but the net consumption was zero, and moreover numerous ene-diols were just as effective on a molar basis. La Du and Greenberg considered that ascorbic acid participates in a cyclic oxidation-reduction and happens to be a substance of the correct oxidation-reduction potential either to participate directly or to protect some other participant. 

Systems which oxidize p-hydroxyphenylpyruvate have been found in hog liver (224,239), rabbit liver (766), rat liver (429,465,810) and dog liver (466,838). Soluble, mitochondria-free p-hydroxyphenylpyruvate oxidase from rat liver oxidizes 2,5-dihydroxyphenylpyruvic acid to acetoacetate at a rate comparable with that of the over-all oxidation of L-tyrosine (429), but will not oxidize 2,5-dihydroxyphenyl-alanine, gentisic acid, or the lactone of 2,5-dihydroxyphenylpyruvic acid (429). A purified rabbit liver oxidase transforms p-hydroxy-... 

Uchida et al. excluded the possible function of ascorbic acid as a peroxide source and considered their enzyme to be an oxidase, not a peroxidase. Their second enzyme converted dihydroxyphenylpyruvic acid to homo-... 

As an example, the reduction of dihydroxyphenylpyruvic acid (DHPP) to dihydrox-yphenyllacetic acid (DHPL), a precursor of rosmarinic acid, is presented in Eq. (49) [mi. 

In Sect. 7.4.3, the reduction of dihydroxyphenylpyruvic acid (DHPP) to dihydrox-yphenyllactic acid (DHPL) was used as an example for discussion of the kinetics of multiple enzyme systems (Eq. (49)). The rate equations for the reduction reaction of DHPP to DHPL (v>i) and the regeneration of PEG-NAD+ to PEG-NADH (v2) have been introduced (Eqs. (50) and (51)). 

An enzyme that synthesises norlaudanosoline (25) has been isolated, and purified, from several plant species which normally produce isoquinoline alkaloids. Substrates for the enzyme were dopamine (21) and, most surprisingly, 3,4-dihydroxyphenylacet-aldehyde (23), and not 3,4-dihydroxyphenylpyruvic acid (22). 4-Hydroxyphenylacetaldehyde was a substrate for the enzyme but not 4-hydroxyphenylpyruvic acid or phenylpyruvic acid. The product of the clearly enzyme-catalysed reaction between (21) and (23) was norlaudanosoline (25) [predominantly the (S)-isomer]. No doubt... 

If the amino-acid (68) is a biosynthetic intermediate it clearly arises in vivo from dopamine and 3,4-dihydroxyphenylpyruvic acid (67). The latter compound is derived from dopa by transamination and the conversion of dopa into (68) thus involves competing processes of decarboxylation and transamination. In P. orientale the former process was apparently favoured seven-fold over transamination whereas in P. somniferum [2- " C]dopa was only incorporated into morphine via dopamine. An explanation of the latter result is that the dopa fed fails to penetrate to the site of the appropriate transaminase in this plant. 

The first approach to 5,6-dihydroxyindole-2-carboxylic acid 2 and related derivatives was due to Beer and co-workers (49JCS2061) and was based on the reductive cyclization of 2-nitro-4,5-dihydroxyphenylpyruvic acid or a derivative. The critical reduction step is achieved with Fe powder in ethanol/acetic acid. Notably, indole 2 was reported to oxidize poorly and only slowly, giving in the course of 24 h a dark-brown solution but not an insoluble product of the melanin type. This observation led to the conclusion that ... it is unlikely that 5,6-dihydroxyindole-2-carboxylic acid is a precursor of melanin . This conclusion was disproved by subsequent studies (97T8281). In the same paper, the preparations and properties of... 

An early key intermediate in benzylisoquinoline biosynthesis is (57), which by decarboxylation affords (59) this in turn leads to (61) and on to alkaloids (Scheme 2). Confirmation of this pathway has come from a study using cell-free preparations of P. somniferum stems and seed capsules. It was found that this preparation catalysed the formation of (57), (59), and (61) from dopamine (54) plus 3,4-dihydroxyphenylpyruvic acid (55) without the addition of 5-adenosyl-methionine, NADPH, and pyridoxal phosphate, the reaction stopped at (57). The formation of the alkaloids reticuline, thebaine, codeine, and morphine, produced by whole plants, could not be detected with this cell-free system. The results confirm not only the intermediacy of (57) and (59) in benzylisoquinoline biosynthesis, but also the involvement of (54) and (55). 

It is not surprising that variations in the ratio of incorporation of the two units of tyrosine were found when the experiments were carried out under different conditions (222). It is evident that several factors in the plant must affect the concentrations of labeled dopamine (CLIII) and 3,4-dihydroxyphenylpyruvic acid (CLIV) derived from the original tyrosine, and whose condensation leads to the benzylisoquinoline structure. 

Dopamine and 3,4-dihydroxyphenylpyruvic acid were added to this cell-free extract, and the formation of labelled (67), (68), and (69) was observed (carrier compounds were added at the end of the incubation). It was stated that no norlaudanosoline-1-carboxylic acid was formed under the incubation conditions when boiled enzyme was used. In none of these experiments was the chirality of the norlaudanosoline-l-carboxylic acid determined. If the reaction yielding this compound is controlled by an enz3une, one would expect this amino-acid to be optically active. The enz3une isolated by Zenk and his co-workers failed to catalyze a reaction between dopamine and 3,4-dihydroxyphenylpyruvic acid. 4-Hydroxy-phenylacetaldehyde did serve as a substrate for the enzyme. To summarize, I believe that the evidence favoring the intermediacy of norlaudanosoline-l-carboxylic acid is somewhat flimsy. It is of course possible that there are two independent routes to nor-laudanosoline, and this is the most charitable explanation of these conflicting results. 

Norlaudanosoline (3,4-dihydroxyphenylpyruvic acid did not serve as a substrate for this enzyme). 

Dopamine was produced by incubating liver microsomes of rabbits with p- and m-tyramine, noradrenaline, and normetanephrine by incubation with w-octopamine, and adrenaline by incubation with p- and w-methyloctop-amine/" The injection of labelled tyramine as well as of labelled octopamine in the intact animal (rat) caused the appearance of labelled noradrenaline and normetanephrine in the urine/ As tyramine is also hydroxylated to octopamine, it is possible that the production of noradrenaline from tyramine not only takes place via its conversion to dopamine, but also via its conversion to octopamine. The possibility that octopamine is produced by the hydroxylation of tyrosine to hydroxyphenylserine with ensuing decarboxylation is under discussion. The production of noradrenaline from 3,4-dihydroxyphenylserine, 192-201) amino-acid so far not discovered in the mammal could be demonstrated both in organ extracts and in intact animals. Finally, a transamination of 3-hydroxy- or 3,4-dihydroxyphenylpyruvate to the corresponding amino-acids (m-tyrosin, dopa), and their decarboxylation to m-tyramine and dopamine was observed in intact animals (cats). For the time being it is impossible to determine the importance of the means of formation of catecholamines which have been referred to here. Of the above-mentioned precursor substances, p- and m-tyramine,( octopamine, ... 

Dihydroxyphenylpyruvate was not oxidized to homogentisate, thus it cannot be an intermediate in the oxidation of p-hydroxyphenyl-pyruvate. The results suggest that the hydroxylation shift of the side chain and decarboxylation of the p-hydroxyphenylpyruvate are simultaneous processes. Additional evidence that 2,5-dihydroxyphenylpyruvate is not the intermediate was obtained by experiments in which the relative rates of oxidation of this compound and of p-hydroxyphenylpyruvate were compared in homogenates of rat liver, where the reaction proceeded to formation of acetoacetic acid. Oxidation of p-hydroxyphenylpyruvate proceeded much more rapidly. Other analogs of p-hydroxyphenylpyruvate were found to be inactive as substrates. 

On the other hand, an enzyme system purified 100-fold from hog liver oxidizes p-hydroxyphenylpyruvate but not 2,5-dihydroxyphenylpyruvate (224). The corresponding purified dog liver oxidase will not oxidize 2,5-dihydroxyphenylpyruvate, nor will it attack o-hydroxyphenylpyruvate, 7 i-hydroxyphenylacetate, p-hydroxyphen-ylacetone, or the lactone of 2,5-dihydroxyphenylp3rruvic acid (467). Furthermore, one molecule of Os is consumed per molecule of p-hydroxyphenylpyruvate and per molecule of COs evolved. These ratios are 1 throughout the course of the oxidation no accumulation of intermediate can be detected by this means (469,470). 

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