Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.     

ON THE INACTIVATION OF ASCORBIC ACID OXIDASE

1. In the absence of protective agents, highly purified ascorbic acid oxidase is rapidly inactivated during the enzymatic oxidation of ascorbic acid under optimum experimental conditions. This inactivation, called reaction inactivation to distinguish it from the loss in enzyme activity that frequently occurs in diluted solutions of the oxidase prior to the reaction, is indicated by incomplete oxidation of the ascorbic acid as measured by oxygen uptake; i.e., "inactivation totals." 2. A minor portion of the reaction inactivation appears to be due to environmental factors such as rate of shaking of the manometers, pH of the system, substrate concentration, and oxidase concentration. The presence of inert protein (gelatin) in the system ameliorates the environmental inactivation to a considerable extent, and variation of the above factors in the presence of gelatin has much less effect on the inactivation totals than in the absence of gelatin. 3. A major portion of the reaction inactivation of the oxidase appears to be due to some factor inherent in the ascorbic acid-ascorbic acid oxidase-oxygen system, possibly a highly reactive "redox" form of oxygen other than H₂O₂ or H₂O. The inactivation cannot be attributed to dehydroascorbic acid, the oxidation product of ascorbic acid. 4. Small amounts of native catalase, native peroxidase, native or denatured methemoglobin, and hemin when added to the system, markedly protect the oxidase against inactivation. Cytochrome c has no such protective action. Likewise proteins such as egg albumin, gelatin, denatured catalase, or denatured peroxidase show no such protective action. 5. None of the protective agents mentioned above affect the initial rate of oxygen uptake or change the total oxygen absorbed for complete oxidation of the ascorbic acid, and hence do not act by removal of hydrogen peroxide, per se. 6. Sodium azide and hydroxylamine hydrochloride which inhibit catalase and peroxidase activity also inhibit the protective action of these iron-porphyrin enzymes.     

A monophenol oxidase activity in extracts of sorghum

A p-hydroxycinnamic acid oxidase activity was present in enzyme preparations from first internodes of Sorghum vulgare variety Wheatland milo when incubated in phosphate buffer at pH 7.5. This preparation had no classical polyphenolase activity but had both peroxidase and catalase activities. Since horseradish preparations catalyzed the same reaction, the oxidation probably is another example of a peroxidase-oxidase reaction. A second substrate was p-hydroxyphenylpyruvic acid. Ferulic acid was slightly active at low concentrations and inhibitory at higher ones. Diphenols such as caffeic and chlorogenic acids were inactive and inhibitory to p-hydroxycinnamic acid oxidation. A variety of monophenols such as tyrosine and cinnamic acid were inactive. An active substrate must have a free monophenolic group and para to this a C₃ side chain with a double bond and probably a free terminal acid group. A sulfhydryl reducing agent at the 5 millimolar level such as mercaptoethanol, reduced glutathione, or dithiothreitol was obligatory. Products were varied and were found in both the ethyl acetate-soluble and insoluble fractions after acidification of the incubation mixtures. With internode extracts, about 1 micromole of O₂ was consumed per micromole of p-hydroxycinnamic acid that disappeared in the presence of mercaptoethanol. Tetrahydrafolic acid plus mercaptoethanol were required for a second step oxidation or a parallel reaction; about 2 micromoles of O₂ were consumed per micromole of p-hydroxycinnamic acid that disappeared. Potassium cyanide, diethyldithiocarbamate, ascorbic acid, and ethylenediaminetetraacetate were inhibitory. A similar mercaptoethanol-dependent monophenol oxidase was present in preparations from green shoots that also contained a classical polyphenolase activity. The activity was present in both soluble and particulate (500 to 100,000 gravity) fractions of internodes. Preliminary studies were made of enzyme complexes in the particulate fractions capable of converting phenylalanine and tyrosine to the level of ferulic acid when the above p-hydroxycinnamic acid oxidase was blocked with ascorbic acid. The ratelimiting step was the hydroxylation of p-hydroxycinnamic acid.     

Mono- and diphenolase activity from fruit of Malus pumila

Apple fruit used for beverage production has a polyphenol oxidase which does not hydroxylate tyrosine under any conditions but it hydroxylates p-coumaric acid in the presence of NADH, and phloridzin in the absence of cofactors. The apparent K_ms for hydroxylation of phloridzin and p-coumaric acid are 1.5 and 4 mM, respectively. However, subsequent oxidation of 3-hydroxyphloridzin or caffeic acid has an apparent K_m of 200 nM. The oxidation products of 3-hydroxyphloridzin are complex and a stable dimeric quinone is finally formed. The apparent K_ms for oxidation of catechin, epicatechin, chlorogenic acid, l-Dopa and 4-methyl catechol are 4.7, 5.7, 6.0, 2.7 and 3.2 mM, respectively. The K_m for oxygen was 4.3 % although there was marked substrate inhibition by oxygen above 30 %. Polyphenol oxidase was stable at pH 3.5–4.5 with an optimum of 4.5.     

Tyrosinase catalyzes an unusual oxidative decarboxylation of 3,4-dihydroxymandelate

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, it catalyzed an unusual oxidative decarboxylation reaction generating 3,4-dihydroxybenzaldehyde as the sole product. The identity of the product was confirmed by high-performance liquid chromatography (HPLC) as well as ultraviolet and infrared spectral studies. None of the following enzymes tested catalyzed the new reaction: galactose oxidase, ceruloplasmin, superoxide dismutase, ascorbate oxidase, dopamine beta-hydroxylase, and peroxidase. Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide, and sodium azide inhibited the reaction drastically, suggesting the participation of the active site copper of the enzyme in the catalysis. Mimosine, a well-known competitive inhibitor of tyrosinase, competitively inhibited the new reaction also. 4-Hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid neither served as substrates nor inhibited the reaction. Putative intermediates such as 3,4-dihydroxybenzyl alcohol and (3,4-dihydroxybenzoyl)formic acid did not accumulate during the reaction. Oxidation to a quinone methide derivative rather than conventional quinone accounts for this unusual oxidative decarboxylation reaction. Earlier from this laboratory, we reported the conversion of 4-alkylcatechols to quinone methides catalyzed by a cuticular phenol oxidase [Sugumaran, M., & Lipke, H. (1983) FEBS Lett. 155, 65-68]. Present studies demonstrate that mushroom tyrosinase will also catalyze quinone methide production with the same active site copper if a suitable substrate such as 3,4-dihydroxymandelic acid is provided.     

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.     

The enzymic oxidation of chlorogenic acid and some reactions of the quinone produced

1. Partially purified preparations of tobacco-leaf o-diphenol oxidase (o-quinol-oxygen oxidoreductase; EC 1.10.3.1) oxidize chlorogenic acid to brown products, absorbing, on average, 1.6atoms of oxygen/mol. oxidized, and evolving a little carbon dioxide. 2. The effect of benzenesulphinic acid on the oxidation suggests that the first stage is the formation of a quinone; the solution does not go brown, oxygen uptake is restricted to 1 atom/mol. oxidized, and a compound is produced whose composition corresponds to that of a sulphone of the quinone derived from chlorogenic acid. 3. Several other compounds that react with quinones affect the oxidation of chlorogenic acid. The colour of the products formed and the oxygen absorbed in their formation suggest that the quinone formed in the oxidation reacts with these compounds in the same way as do simpler quinones. 4. Some compounds that are often used to prevent the oxidation of polyphenols were tested to see if they act by inhibiting o-diphenol oxidase, by reacting with quinone intermediates, or both. 5. Ascorbate inhibits the enzyme and also reduces the quinone. 6. Potassium ethyl xanthate, diethyldithiocarbamate and cysteine inhibit the enzyme to different extents, and also react with the quinone. The nature of the reaction depends on the relative concentrations of inhibitor and chlorogenic acid. Excess of inhibitor prevents the solution from turning brown and restricts oxygen uptake to 1 atom/mol. of chlorogenic acid oxidized; smaller amounts do not prevent browning and slightly increase oxygen uptake. 7. 2-Mercaptobenzothiazole inhibits the enzyme, and also probably reacts with the quinone; inhibited enzyme is reactivated as if the inhibitor is removed as traces of quinone are produced. 8. Thioglycollate and polyvinylpyrrolidone inhibit the enzyme. Thioglycollate probably reduces the quinone to a small extent.     

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Monophenolase activity of avocado polyphenol oxidase

Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l-tyrosine, d-tyrosine, tyramine and p-cresol, and (b) the oxidation of the corresponding o-dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl-6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10^?4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K_m values indicate that tyramine, dopamine, p-cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.     

The mechanism of the periodate–thiobarbituric acid reaction of sialic acids

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).     

Study of the Kinetics of Oxidation of Monophenols by Tyrosinase. The Effect of Reducers

Kinetics of oxidation of monophenols by tyrosinase from the fungus Aspergillus flavipes 56003 and the effect of Fe²⁺, serine, and ascorbic acid on this reaction were studied. The effectors were shown to accelerate the oxidation of monophenols, decreasing the lag-time of the reaction. It is assumed that the activation of the tyrosinase in the presence of Fe²⁺ is due to a direct reduction of the active site copper ions. Serine and ascorbic acid are supposed to affect the reaction of quinone transformation. The activation of the enzyme in the presence of Fe²⁺ suggests that the oxidation of monophenols is an autocatalytic process.     

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.     

The effect of o-diphenols upon the microsomal NADPH and NADH oxidase activities

Catechol and catecholamines have been assayed upon the microsomal NADPH and NADH oxidase activities. Epinephrine shows a catalytic effect on the NADPH oxidation characterized by a small lag. The two to threefold increase in rate can be suppressed by Superoxide dismutase if the enzyme is added before the reaction begins. The catalytic effect is ascribed to a quinone formed by two electron oxidation of epinephrine by the Superoxide ion. The quinone, which is not catalytically active in the NADH chain, appears to mediate electrons between the NADPH-cytochrome c reductase and oxygen. The four electron oxidation product adrenochrome is also active upon the NADPH chain but inactive upon the NADH chain.Epinephrine did not change the menadione-stimulated NADPH oxidase activity. Presumably, during this and the NADH oxidase activities, two electrons are simultaneously transferred to the oxygen molecule.Catechol and catecholamines doubled the rate of autoxidation of NADH in the presence of catalytic amounts of NADH-cytochrome b₅ reductase and cytochrome b₅, a result which suggests Superoxide ion formation in the autoxidation of the cytochrome.Epinephrine does not act upon the desaturation of endogenous substrate or upon endogenous lipid peroxidation.     

Mechanisms of the antilipolytic response of human adipocytes to tyramine,a trace amine present in food

Tyramine is found in foodstuffs, the richest being cheeses, sausages, and wines. Tyramine has been recognized to release catecholamines from nerve endings and to trigger hypertensive reaction. Thereby, tyramine-free diet is recommended for depressed patients treated with irreversible inhibitors of monoamine oxidases (MAO) to limit the risk of hypertension. Tyramine is a substrate of amine oxidases and also an agonist at trace amine-associated receptors. Our aim was to characterize the dose-dependent effects of tyramine on human adipocyte metabolic functions. Lipolytic activity was determined in adipocytes from human subcutaneous abdominal adipose tissue. Glycerol release was increased by a fourfold factor with classical lipolytic agents (1 μM isoprenaline, 1 mM isobutylmethylxanthine) while the amine was ineffective from 0.01 to 100 μM and hardly stimulatory at 1 mM. Tyramine exhibited a partial antilipolytic effect at 100 μM and 1 mM, which was similar to that of insulin but weaker than that obtained with agonists at purinergic A1 receptors, α₂-adrenoceptors, or nicotinic acid receptors. Gi-protein blockade by Pertussis toxin abolished all these antilipolytic responses save that of tyramine. Indeed, tyramine antilipolytic effect was impaired by MAO-A inhibition. Tyramine inhibited protein tyrosine phosphatase activities in a manner sensitive to ascorbic acid and amine oxidase inhibitors. Thus, millimolar tyramine restrained lipolysis via the hydrogen peroxide it generates when oxidized by MAO. Since tyramine plasma levels have been reported to reach 0.2 μM after ingestion of 200 mg tyramine in healthy individuals, the direct effects we observed in vitro on adipocytes could be nutritionally relevant only when the MAO-dependent hepato-intestinal detoxifying system is overpassed.     

Enhancement of antibacterial effects of epigallocatechin gallate, using ascorbic acid

Although plant polyphenols such as (−)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable in solution. Because the instability of polyphenols is attributable to their oxidation, we examined the effects of antioxidants and inhibitors of polyphenol oxidation on the maintenance of polyphenol antibacterial activity. The antibacterial activity of EGCG was enhanced in the presence of ascorbic acid, and ascorbic acid was the most effective for retaining the concentration of stable EGCG. On the other hand, the antibacterial activity of EGCG was lowered in the presence of casein in spite of its suppressing effect on the EGCG decrease. The effect of EGCG on the antibiotic resistance of MRSA was also enhanced in the presence of ascorbic acid. The addition of an antioxidant may affect other pharmacological effects of polyphenols in analogous ways, although this does not mean the clinical usefulness of the addition directly.     

Reaction of azide radicals with amino acids and proteins.

The azide radical N3 reacts selectively with amino acids, in neutral solution preferentially with tryptophan (k (N3 + TrpH) = 4.1 X 10(9) dm3 mol(-1s-1) and in alkaline solution also with cysteine and tyrosine (k(N3 + CyS-) = 2.7 X 10(9) dm3 mol-1s-1) and k(N3 + TyrO-) equals 03.6 X 10(9) dm3 mol-1s-1). Oxidation of the enzyme yADH by N3 involves primary attacks, mainly at tryptophan residues, and subsequent slow secondary reactions. N3-induced inactivation of yADH is likely to occur upon oxidation of tryptophan residues in the substrate binding pocket (58-TrpH and 93-TrpH) since the substrate ethanol although unreactive with N3, protects yADH and since elADH, which does not contain tryptophan in the substrate pocket, is comparatively resistant against N3-attack. N3 exhibits low reactivity with nucleic acid derivatives and it is inert towards aliphatic compounds such as methanol and sodium dodecylsulphate.     

Studies on Tyrosinase in Housefly

Occurrence of protyrosinase in the prepupae of housefly, Musca vicina Maquart, and its activation were described. The prepupae possessing no appreciable tyrosinase activity could be separated from the other aged pupae by putting them into water. Homogenate prepared from the prepupae contains protyrosinase and has no activation system for the proenzyme. As has been reported by Ohnishi²⁾ a certain activator occurred naturally in the aged pupae apparently activates the protyrosinase in vitro. However, contrary to Ohnishi’s results³⁾ it was found that this protyrosinase can be activated by the treatment with sodium dodecyl sulfate in vitro.     

First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast

Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate.     

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.