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Chloroplast-rich particles of sugar beet and Euglena gracilis are able to carry out the light-, H2O2- and O2-independent syntheses of a nonaprenyltoluquinol, an octaprenyltoluquinol and a phytyltoluquinol from homogentisate and nonaprenyl, octaprenyl and phytyl pyrophosphate. The formation of these compounds probably takes place by the concomitant polyprenylation (or phytylation) and non-oxidative decarboxylation of homogentisate.

Photosynthesis experiments with 14 CO 2 established that of 16 Droseraceae species tested Drosophylum lusitanicum incorporated the highest amount of label into plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone). Tyrosine-[β- 14 C] fed to Drosophyllum was shown to label plumbagin efficiently (20% incorporation). Extensive chemical degradation of the labeled naphthoquinone showed, however, that the incorporation of tyrosine was indirect, the label being distributed throughout the molecule. It was established that plumbagin and the closely related 7-methyljuglone are biosynthesized via the acetate-polymalonate pathway. Tyrosine is broken down to acetate in this tissue via the homogentisate pathway, which was demonstrated by feeding and incorporation of label into plumbagin of intermediates such as homogentisate-[ 14 C], maleyl- and fumarylacetoacetate-[ 14 C]. Simultaneous application of tyrosine-[β- 14 C] and α,α′-bipyridyl, an inhibitor of the homogentisate oxigenase, led to an accumulation of homogentisate-[ 14 C] within the tissue. The degradation of tyrosine to acetate by Drosophyllum is not due to epiphytic bacteria since ring cleavage of tyrosine and formation of plumbagin from breakdown products occurred both within sterile grown plants and sterile cell suspension cultures. In tissue kept in darkness, plumbagin undergoes a slow turnover with a half life of about 400 hr.

Two radiochemical methods for the determination of p -hydroxyphenylpyruvate hydroxylase have been developed. The substrate, [U- 14 C] p -hydroxyphenylpyruvate is prepared immediately before use by the action of l -amino acid oxidase on l -[U- 14 C]tyrosine. For the measurement of enzyme activity in liver biopsies around 0.1 mg wet weight of liver is needed. A homogenate of liver tissue is incubated with the substrate and the formed homogentisate quantitated by scanning a thin-layer plate for the distribution of isotope. With this technique the enzyme activity in human liver was 0.34 (0.15–0.63) U/g of wet weight. When high sensitivity is not required a more rapid assay may be used, which is based on the determination of 14 CO 2 formed in the conversion of p -hydroxyphenylpyruvate to homogentisate. With this assay about a hundred determinations can be carried out in one day. During storage at −60° for 2 months the enzyme activity in human liver remains constant.

Two methods for the assay of p -hydroxyphenylpyruvate hydroxylase ( p -hydroxyphenylpyruvate, ascorbate: O 2 oxidoreductase (hydroxylating), EC 1.14.2.2) activity are described. (1) Chemical method: The homogentisate formed was condensed with cysteine to yield a 1,4-thiazine with an absorption maximum at 390 nm. (2) Radiochemical assay: The release of 14 CO 2 from p -hydroxyphenyl[ carboxy - 14 C]-pyruvate was used as a measure of enzyme activity. Enzyme activation by certain reducing agents, pH optimum, tissue distribution, and fetal and adult enzyme activities are reported. Phenylpyruvate and 3,4-dihydroxyphenylpyruvate were substrates of the enzyme and were mutually competitive to p -hydroxyphenylpyruvate oxidation. The substrates, p -hydroxyphenylpyruvate and oxygen, are inhibitory at high concentration. Hydrogen peroxide, generated by the substrate and oxygen, may account for the observed substrate inhibition. The enzyme is found only in liver and kidney. The significance of these findings to phenylketonuria and tyrosinemia is discussed.

A study of the diurnal variation of the activities of the first three enzymes of the tyrosine catabolic pathway in rat liver was carried out. Tyrosine α-ketoglutarate transaminase (TKT), as had been shown previously, exhibited a large diurnal variation in activity. The second enzyme, p-hydroxyphenylpyruvate hydroxylase also had a diurnal variation which qualitatively paralleled that of TKT, although of a much lower amplitude. The third enzyme, homogentisate oxygenase did not exhibit change in its activity. Further studies were carried out on the induction of these three enzymes by tyrosine. It was found that in the adrenalectomized animal tyrosine injection produced an increase in p-hydroxyphenylpyruvate activity to the same extent as was observed during the diurnal rhythm studies.

The specific activities (per milligram of DNA) of the three rat liver sequential enzymes—tyrosine aminotransferase (TAT), p-hydroxyphenyl pyruvate hydroxylase (PHPP hydroxylase), and homogentisate oxygenase develop coordinately in a stepwise pattern from birth to adulthood. Just after birth about 80% of TAT but only 20–30% of PHPP hydroxylase appears to be active in vivo. The active fraction of TAT remains essentially constant during development of the rat but that of PHPP hydroxylase increases with age to reach about 90% in adulthood. The differences in the enzymes of the infant and adult rat (ontogenic changes) are, in general, similar to those observed in the enzymes, respectively, of reptile and mammal (phylogenic changes). The biochemical mechanisms involved in the in vivo activation of PHPP hydroxylase and the in vitro reactivation of the adult enzyme inactivated by various reagents are analyzed.

SummaryThe activity of phenylalanine hydroxylase from rat liver can be inhibited in vitro by several organic acids including a few catabolites of phenylalanine, e.g., phenyl-pyruvate and homogentisate. However, the concentrations of such compounds necessary to produce significant inhibition of a generally competitive nature is far in excess of physiological amounts, and the effects can be minimized when an adequate amount of pteridine cofactor is present. Thus, the lowered activity of hydroxylase measured in experimental phenylketonuria may be attributed to a real suppression of enzyme synthesis, rather than inhibition by catabolites.

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by ω-oxidation of the terminal methyl group, side-chain β-oxidation to 4-phenylbutyrate, both β- and α-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.

The major pathway for tyrosine catabolism in the animal is the non-oxidative transamination of tyrosine to p-hydroxyphenylpyruvate and its subsequent oxidation to homogentisate. The homogentisate is oxidized to fumarylacetoacetate (maleylacetoacetate), which is finally hydrolyzed to fumarate (malate) and acetoacetate. The enzyme system responsible for this catabolism, tentatively called the tyrosine oxidizing system, is soluble and a partial purification can be accomplished with high speed centrifugation of homogenates of liver (1) and kidney (2). When these preparations are used it has been observed (3) that one mole of tyrosine will be completely oxidized to one mole of acetoacetate and one mole of fumarate, with the utilization of two moles of oxygen. In order to obtain maximal tyrosine

Publisher Summary This chapter discusses the coenzyme function of glutathione in cis-trans isomerization. The cis and trans isomerization of maleyl- to fumaryl-acetoacetate is catalyzed by a liver enzyme that requires glutathione (GSH) as its co-enzyme. There are indications that geometric isomerizations must also occur in the metabolism of vitamin A and benzene. The nature of the reaction is also different from the one other enzyme reaction in which GSH plays a coenzyme role. Maleyl-acetoacetate is formed by the oxidative ring opening of homogentisate in the system of soluble liver enzymes oxidizing tyrosine to acetoacetate and fumarate. On addition of glutathione to the mixed liver enzymes, the isomerase present converts maleyl- to fumaryl-acetoacetate and the latter can then be hydrolyzed.

Summary1. The oxidation by bacteria of salicylate, gentisate, and 2:3-dihydroxyben-zoate has been examined with the technic of simultaneous adaptation. 2. Catechol appears to be an intermediate in the oxidation of salicylate. 3. Organisms grown on gentisate and 2:3-dihydroxybenzoate show no adaptation to any likely known intermediates. 4. It is suggested that the oxidation of gentisate may be analogous to that of homogentisate, with formation of a straight-chain β-diketo-dicarboxylic acid.

1. 1. A tyrosine-catabolizing system similar to that found in rat liver was found to be present in Tetrahymena pyriformis , strain W. The five constituent enzymes of the system, i.e. tyrosine aminotransferase, p -hydroxyphenylpyruvate oxidase, homogentisate oxidase, maleylacetoacetate isomerase and fumarylacetoacetate hydrolase were assayed in extracts of the cells. Fumarylacetoacetic acid was prepared enzymatically from homogentisic acid with a protein fraction of the cells. 2. 2. All the tyrosine-catabolizing enzymes and phosphoenolpyruvate (PEP) carboxykinase were repressed by glucose and by acetate. The first enzyme of the catabolic sequence, tyrosine aminotransferase, and PEP carboxylase were repressed by glucose in both logarithmic and stationary phases of growth while the four subsequenct catabolis enzymes were repressed essentially only in stationary phase. A close metabolis relationship was found to exist between tyrosine aminotransferase and PEP carboxykinase. 3. 3. The repression of enzymes by glucose and acetate was accompanied by a repression of carbon flow from tyrosine to glycogen. 4. 4. An inverse relationship exists between the activities of tyrosine aminotransferase and PEP carboxykinase on the one hand and isocitrate lyase on the other. This relationship and the repression of the catabolic enzymes by acetate, a glucogenic precursor in Tetrahymena , suggests that the regulation of the tyrosine-catabolizing enzymes and particularly of tyrosine aminotransferase is geared to the demand for gluconeogenesis from tyrosine in these cells.

Excerpt Ochronosis is usually associated with the rare hereditary metabolic disease, alcaptonuria, in which there is a defect in the hepatic enzyme, homogentisic acid oxidase, and consequently an a...

An experimental alkaptonuria induced by tryptophan deficiency has been observed in the rat. The condition of alkaptonuria followed development of signs of tryptophan deficiency as judged by weight loss of the young, growing rat and its general appearance. Homogentisic acid was identified by a comparison of its behavior on thin-layer chromatography with authentic homogentisate and by means of the enzyme homogentisic acid oxidase. The condition was relieved by the return of tryptophan to the diet.

An autopsy case of alkaptonuria in a 47-year-old man is reported. Since childhood, he had noticed black color of his excreted urine. Autopsy revealed characteristic pathologic changes due to alkaptonuria such as ochronosis and ochronotic spondylosis, etc. The primary cause of death was pontine hemorrhage. Histologically, two types of ochronotic pigment were observed. Enzyme activity of homogentisate oxygenase in the liver and kidney homogenate was extremely decreased.

Publisher Summary This chapter discusses the assay, purification, and properties of homogentisate oxygenase. It can be assayed by two methods—namely, by measuring O 2 consumption manometrically and by measuring 4-maleylacetoacetate spectrophotometrically at 330 m μ . The latter method cannot be used for assaying crude extracts, because they accumulate no maleylacetoacetate. A unit of enzyme activity is the amount of enzyme that consumes 1 micromole of oxygen per minute with the manometric method or produces 1 micromole of maleylacetoacetate per minute with the spectrophotometric method. Specific activity is defined as units of activity per milligram of protein. This enzyme shows maximal activity at about pH 6.0. At pH 6.0–7.2, the enzyme can be stored for several weeks at -20° or several days in a refrigerator without the loss of activity. The enzyme is sensitive to oxygen and easily inactivated by aeration. No activity can be detected in the absence of ferrous ion. This enzyme is inhibited by 85% by 2 × 10 -4 M p -chloromercuribenzoate.

Alkaptonuria (AKU) is a rare inherited genetic disorder of phenylalanine and tyrosine metabolism. AKU represents an autosomal recessive condition caused by a defect in the enzyme homogentisate 1,2-dioxygenase, which participates in the degradation of tyrosine. As a result, homogentisic acid and its oxide, accumulate in the blood and are excreted in urine in large amounts. The polymer of homogentisc acid called alkapton, impregnates bradotrophic tissues such as cartilage. A clinical trial aimed at proving efficacy of nitisinone in AKU is currently underway.

Ochronosis is a rare inborn error of metabolism caused by a lack or deficiency of the enzyme homogentisic acid oxidase. An additional case has herein been reported exhibiting the classic changes in the sclerae, urine and joints. Ochronosis es rar. Illo es un innate error metabolic causate per manco o carentia del enzyma oxydase de acido homogentisic. Un caso additional es hic reportate. Illo exhibi le alterationes classic in le scleras, le urina, e le articulationes.

Publisher Summary This chapter discusses the enzymes involved in conversion of tyrosine to acetoacetate and describes the determination of L-tyrosine-oxidizing system of liver, tyrosine-glutamic acid transaminase, p -hydroxyphenylpyruvate enol-keto tautomerase, homogentisate oxidase from rat liver, maleylacetoacetate isomerase, and fumarylacetoacetate hydrolase. In the process of determination of L-tyrosine-oxidizing system of liver, the several enzymes catalyzing the oxidation of L-tyrosine to acetoacetate are present in the soluble fraction of rat liver. The overall system can be most easily demonstrated with the preparation of Knox and LeMay-Knox, using oxygen uptake as the index of activity. Partial oxidation with accumulation of intermediate compounds often results in other types of preparations, and this can be detected only by determination of the total oxygen uptake with tyrosine or by measurement of the final products. In the process of determination of p-hydroxyphenylpyruvate enol-keto tautomerase, The enol of p-hydroxyphenylpyruvate absorbs intensely at 300 mμ, whereas the keto form does not absorb significantly in the near-ultraviolet region. The equilibrium mixture is predominantly the keto form, but the enol can be stabilized in borate, presumably by the formation of a complex similar to those formed by other α-hydroxy acids. Equilibrium mixtures in borate consist of nearly equal amounts of both forms, and the enzyme can be assayed in borate spectrophotometrically by following the approach to this equilibrium, starting with either form. The keto form is usually used, since its solutions are relatively stable.

Summary Homogentisate oxygenase from Pseudomonas ftuorescens adapted to tyrosine has been isolated, purified and crystallized. The crystallized enzyme was homogeneous on ultracentrifugation, and its sedimentation constant was found to be 11.8 S. The molecular weight of the enzyme was calculated to be about 380 000. Like the mammalian liver enzyme, bacterial homogentisate oxygenase requires ferrous iron as a cofactor. For maximal activity, the enzyme requires at least 10 min preincubation with ferrous iron at pH 6.0. The optimal pH is 6.0. Both glutathione and ascorbate are also required for maximal activity at pH 6.0, but only the former is essential at pH 5.4. The Km values are 6 · 10−4 M for homogentisate and 1 · 10−4 M for Fe2+ at pH 6.0. Unlike the mammalian liver enzyme, the bacterial enzyme is fairly stable on aging or storage. p-Chloromercuribenzoate inhibits the enzyme with respect to ferrous iron, but non-competitively with respect to homogentisate. The properties of bacterial homogentisate oxygenase are discussed in comparison with those of the mammalian liver enzyme.

The tyrosine metabolites, p- hydroxyphenylpyruvate , homogentisate, and fumarylacetoacetate were examined as inhibitors and inactivators of purified rat liver tyrosine α-ketoglutarate transaminase. Of these, homogentisate proved to be a very powerful inactivator when the enzyme was preincubated with it. This effect was blocked by prior addition of pyridoxal phosphate, α-ketoglutarate and dithiothreitol, but could not be reversed by these compounds. The homogentisate inactivated enzyme retained its ability to interact with the antibody to native tyrosine α-ketoglutarate transaminase.

SummaryAlcaptonuric urines, as well as solutions of homo-gentisic acid or lead homogentisate, give off light when made alkaline and shaken with air.