Kinetic analysis of catechin oxidation by polyphenol oxidase at neutral pH

Catechin oxidation by peach polyphenol oxidase was performed in a pH range of 3.5-8.0. At acidic pH, maximal spectral changes were observed at 390nm and at pH 7.5, at 430nm. Catechin oxidation was studied at pH 7.5 to avoid the formation of free radicals. The results obtained allowed us to propose a pathway for the enzymatic oxidation of catechin, according to which enzymatic oxidation produces the corresponding catechin-o-quinone, which suffers the nucleophilic attack of another catechin unit, leading to the formation of a dimer. This dimer is then oxidized by the enzymatically generated o-quinone. The progress curves obtained for catechin oxidation by PPO showed a lag period, whose length changed with enzyme and substrate concentrations, and which must have been caused by the chemical reactions taking place after the enzymatic reaction. The results obtained by simulation of the model produced the same qualitative dependences as obtained experimentally.     

Comparison of substrate specificity of tyrosinases from Trichoderma reesei and Agaricus bisporus

Understanding the substrate specificity of tyrosinases (EC 1.14.18.1) as well as their capability to oxidize peptide-bound tyrosine residues is important in a view of applicability of tyrosinases. In the present study, two fungal tyrosinases, an extracellular enzyme from the filamentous fungus Trichoderma reesei (TrT) and an intracellular enzyme from the edible mushroom Agaricus bisporus (AbT) were compared. Oxidation of various mono- and diphenolic compounds and tyrosine-containing tripeptides was examined and kinetic constants determined using spectrophotometric and oxygen consumption measurements. TrT and AbT were found to show notable differences in their substrate specificity. TrT generally showed 10-fold higher K_m values than AbT. The presence of a carboxylic and amine group in the substrate influenced the enzymes differently. While the substrates with a carboxyl group were observed not to be effectively oxidized by AbT, the amine group seemed to hider the oxidation in the TrT-catalyzed reactions. Moreover, the UV–visible absorption spectra on the oxidation of catechol and hydrocaffeic acid showed that the product patterns were different between the enzymes. The result is interesting as the primary products from tyrosinase-catalyzed reactions were assumed to be identical with both enzymes. Furthermore, a nucleophilic 3-methyl-2-benzothiazolinone hydrazone (MBTH) affected differently on the activity of the tyrosinases: the lag period related to the oxidation of monophenols was prolonged by MBTH with TrT, whereas with AbT the lag was shortened.     

Kinetic mechanism and product characterization of the enzymatic peroxidation of pterostilbene as model of the detoxification process of stilbene-type phytoalexins

The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by peroxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the enzymatic pool of pathogens. Kinetic characterization of the pterostilbene oxidation reaction pointed to an optimum pH of 7.0, at which value the thermal stability of POX was studied. Moreover, the data showed that pterostilbene inhibits POX activity at high concentrations of substrate. Several kinetic parameters, including V_max, K_m and K_I, were calculated and values of 0.16 ΔAbs min⁻¹, 14.61 μM, and 31.41 μM were reported. To understand the possible physiological role of this reaction in the phytoalexin detoxification process, the products of pterostilbene oxidation were identified using HPLC-MS and a radical-radical coupling reaction mechanism was proposed. Three main products with a high molecular weight and pronounced hydrophobicity were identified: pterostilbene cis dehydromer, pterostilbene trans dehydromer and pterostilbene open dimer. The dimeric structures of these molecules indicate that the pterostilbene oxidation reaction took place at the 4′-OH position of the hydroxystilbenic moieties and the three above mentioned dimeric products were found, due to the ability of electron-delocalized radicals to couple at various sites. Finally, the capacity of cyclodextrins (CDs) as starch model molecules in plants to complex both the substrate and the products of the oxidation reaction was evaluated. The inhibition process of POX activity was modified at high pterostilbene concentrations due to sequestering of the substrate reaction and to the different affinity of the reaction products for CDs.     

Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray‐ionization mass spectrometry

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β‐alanyl‐l ‐histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray‐ionization time‐of‐flight mass spectrometry (ESI‐TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI‐TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N‐terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI‐TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time‐dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI‐TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.     

Formation of homovanillic acid dimer by enzymatic or Fenton system - catalyzed oxidation

Homovanillic acid is the most extensively employed reagent for the fluorometric detection of peroxidase. However, the assays based on the determination of the oxidation product of homovanillic acid do not allow a selective detection of the enzyme, because chemical or physical factors can interfere with the fluorometric determination. The aim of this work was to verify if other enzymatic or non-enzymatic systems might catalyze the homovanillic acid oxidation. The reaction was investigated by spectrophotometric and fluorometric assays; HPLC analysis was used to separate homovanillic acid from its oxidation product and to obtain information on the oxidation process. The results obtained showed that soybean lipoxygenase in the presence of hydrogen peroxide can oxidize homovanillic acid with the formation, by an o,o'-biphenyl linkage, of the corresponding dimer as the sole reaction product. The reaction followed Michaelis-Menten kinetics, for both homovanillic acid and hydrogen peroxide. Other systems, such as cytochrome c/H(2)O(2) and Fenton reagents, were also able to oxidize homovanillic acid to its dimer. It can be affirmed that possible interference by other oxidative systems - that could be present in the biological materials tested - should be considered in assays of peroxidase activity based on the detection of the dimer of homovanillic acid.     

A new enzymo-chemical method for simultaneous assay of methanol and formaldehyde

A new enzymo-chemical method for the simultaneous assay of methanol and formaldehyde in mixtures is described which exploits alcohol oxidase (AO) and aldehyde-selective reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme is used for methanol oxidation to formaldehyde and MBTH plays a double role: 1) at the first step of reaction, it forms a colorless azine adduct with pre-existing and enzymatically formed formaldehyde and masks it from oxidation by AO; 2) at the second step of reaction, non-enzymatic oxidation of azine product to cyanine dye occurs in the presence of ferric ions in acid medium. Pre-existing formaldehyde content is assayed by colorimetric reaction with MBTH without treating samples by AO, and methanol content is determined by a gain in a colored product due to methanol-oxidising reaction. Possibility of differential assay of methanol and formaldehyde by the proposed method has been proved for model solutions as well as for real samples of industrial waste and technical formaline. A threshold sensitivity of the assay method for both analytes is near 1 microM that responds to 30-32 ng analyte in 1 ml of reaction mixture and is 3.2-fold higher when compared to the chemical method with the use of permanganate and chromotropic acid. Linearity of the calibration curve is reliable (p < 0.0001) and standard deviation for parallel measurements for real samples does not exceed 7%. The proposed method, in contrast to the standard chemical approach, does not need the use of aggressive chemicals (concentrated sulfuric, phosphoric, chromotropic acids, permanganate), it is more simple in fulfillment and can be used for industrial wastes control and certification of formaline-contained stuffs.     

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.     

Catabolic Activities of Neisseria meningitidis: Utilization of Glutamate

Glutamate was catabolized at a rapid rate by Neisseria meningitidis, group B. Surprisingly, there was a lag of 5 to 30 min in respiration, but not in CO(2) production from C(1), and an appreciable amount of succinate accumulated. The eventual rapid rate of respiration was not prevented by the addition of chloramphenicol. The lag period was eliminated by combinations of substrates that favored the activity of a glutamate-oxaloacetate transaminase. It is suggested that with glutamate as the sole substrate, the reaction terminated at succinate, required only moderate O(2) uptake, and did not result in the transport of succinate to enzymatic sites. The lag period represented the time required for the accumulation of succinate and its transport to enzymatic sites by energy provided by the metabolism of the remaining glutamate. When the transaminase was operative, on the other hand, successive products of the reaction were immediately placed in contact with enzymatic sites.     

Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms

Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of ~88 and ~87kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III)OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.     

The oxidation of NN-dimethyl-p-phenylenediamine by oxidizing agents and by caeruloplasmin

1. The oxidation of NN-dimethyl-p-phenylenediamine (DPD) by inorganic oxidants and by caeruloplasmin was studied. Some experiments were also made with NNN'N'-tetramethyl-p-phenylenediamine (TPD). 2. E(mM) (550) of the first free radical oxidation product of DPD (DPD(+)) was 9.8 and E(mM) (563) of the corresponding product of TPD (TPD(+)) was 12.5. 3. The non-enzymic decomposition of DPD(+) was studied with respect to temperature, pH, concentration and DPD/DPD(+) ratio, thus defining conditions for enzyme experiments under which DPD(+) extinction at 550mmu was proportional to enzyme activity. 4. Rates of oxidation of DPD to DPD(+) by caeruloplasmin were constant over a range of DPD concentrations. At low DPD concentrations a lag period occurred, which was eliminated by addition of DPD(+). 5. A lag period was not observed with TPD, but at low TPD concentrations the rate of TPD(+) formation was greater when TPD(+) was added. This suggests that TPD(+) may compete weakly as a substrate with TPD and may be oxidized further by the enzyme before a non-enzymic reaction with TPD to form more TPD(+). 6. With DPD sulphate or acetate or TPD sulphate as substrate, Lineweaver-Burk plots were curved. With DPD hydrochloride the chloride ion caused inhibition at higher concentrations, opposing the curvature. 7. Curved Lineweaver-Burk plots were interpreted in terms of two types of substrate binding site with different K(m) values but similar V(max.) values. 8. The apparent thermodynamic changes associated with enzyme-substrate-complex formation at the sites with higher K(m) suggest that considerable conformational change may occur on binding at these sites. 9. With substrate concentrations at which only the low-K(m) sites are involved 2mol. of DPD(+)/mol. of caeruloplasmin are formed before a steady state is established. At higher substrate concentrations up to 3.2mol. of DPD(+)/mol. of caeruloplasmin are formed at this initial stage. 10. Results are discussed in relation to caeruloplasmin structures in which (a) two valence-changing and two permanently cuprous copper atoms are more accessible than the remaining four copper atoms or (b) binding of substrate at one site hinders access of substrate to another site.     

Eriodictyol and naringenin inhibit the formation of AGEs: An in vitro and molecular interaction study

Maillard reaction occurs between the carbonyl group of reducing sugars and the free amino groups of protein, which eventually results in the formation and accumulation of advanced glycation end products (AGEs) irreversibly. Excessive production of AGEs is associated with many diseases, such as Alzheimer disease, neuropathy, retinopathy, and nephropathy. In this study, the effects of eriodictyol and naringenin on the inhibition of AGEs were studied with bovine serum albumin (BSA)–methylglyoxal (MGO) model by spectroscopic techniques and molecular docking methods. The fluorescence spectroscopy results suggested that eriodictyol and naringenin could inhibit the formation of AGEs. Circular dichroism (CD) studies indicated that eriodictyol and naringenin could stabilize the structure of BSA and inhibit the formation of AGEs. The molecular docking results demonstrated that eriodictyol formed two hydrogen bonds with Lys 350 and Leu 480 and the main forces were hydrogen bonding and hydrophobic interactions. However, naringenin interacted with Arg 484 of BSA, and the main force was hydrophobic interaction. It can be concluded that eriodictyol and naringenin can inhibit the formation of AGEs and eriodictyol has stronger inhibitory activity of AGEs than that of naringenin, which is probably due to the additional hydroxyl group in the position C‐3′ of B ring of eriodictyol.     

Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium.

The fatty acid (omega-2) hydroxylase from Bacillus megaterium ATCC 14581 was examined with respect to some general enzymatic properties attributed to an intact complex isolated in a partially purified state. Hydroxylase specific activity was found to increase with increasing protein concentration in a manner consistent with a reversible association of the components in the complex. There was a substantial kinetic lag phase for palmitate hydroxylation which was abolished by a substrate preincubation in the absence of NADPH. The substrate bound and presumably activated the hydroxylase complex without the formation of a substrate-derived intermediated. The oxidation of NADPH and the hydroxylation of palmitate were found to occur in a one to one molar ration, independent of the protein concentration. Finally, a cytochrome P-450 component of the complex was identified on the basis of its CO-binding difference spectrum. It appears, that this cytochrome P-450 component is not identical to P-450 meg of the steroid hydroxylase system of B. megaterium ATCC 13368, since progesterone, an active substrate for the latter, is not hydroxylated by the preparation from B. megaterium ATCC 14581.     

Enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.

Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10 <> p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpTT). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpT<>dT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpT<>dT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.     

Slow association-dissociation equilibrium of NADP-linked isocitrate dehydrogenase from beef liver in relation to catalytic activity.

In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP-linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer-dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a 'hysteretic' behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.     

Horseradish peroxidase-catalyzed oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols

The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H₂O₂ as oxidative agent. The findings indicate that enzyme (H₂O₂ oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compounds. On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the observed increase in the absorbance at 502 nm (8,355 cm^{−1 −1} of extinction molar coefficient) due to the formation of a red azo dye compound resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system, HRP can be determined in picomolar levels by a fixed time method.     

The peroxidase-catalysed oxidation of a chalcone and its possible physiological significance

1. Crystalline horseradish peroxidase catalysed the oxidation of 2',4,4'-trihydroxychalcone (isoliquiritigenin) in the presence of trace amounts of hydrogen peroxide under aerobic conditions. One atom of oxygen was consumed for each molecule of substrate. 2. The reaction course comprised a lag phase and a linear phase. The optimum pH for the linear phase of the reaction was about 7.5. The length of the lag phase decreased with increasing pH. It is suggested that the chalcone anion is the actual substrate for the reaction. 3. No evidence for the production of reducing free radicals or perhydroxyl radicals during the reaction could be found. 4. 4',7-Dihydroxyflavonol and 4',6-dihydroxyaurone were isolated from the reaction mixture. The immediate products of the reaction may have included 3,4',7-trihydroxyflavanone and 4',6-dihydroxy-2-(alpha-hydroxybenzyl)coumaran-one, which can be readily converted non-enzymically into the flavonol and aurone respectively. 5. A similar reaction was catalysed by cell-free extracts of hypocotyls of Phaseolus vulgaris. 6. The physiological significance of the reaction is discussed in terms of a possible free-radical mechanism. An analogy may exist between flavonoid biosynthesis and lignin formation.     

The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

Kinetic studies of L-aspartase from Escherichia coli: substrate activation

The enzyme L-aspartase from Escherichia coli was observed to have a time lag during the production of aspartic acid from fumarate and ammonia. This time lag is pH dependent, with little lag observed below pH 7.0 and a very extensive lag observed above pH 8.0. This time lag was also found to be dependent on both substrate and divalent metal ion concentrations and on the degree of proteolysis of L-aspartase. The observed lag, in the reaction examined in the amination direction, has been found to be correlated with the nonlinear kinetics seen at higher pH in the deamination direction. Both phenomena are consistent with a model in which there is a separate activator site for the substrate, L-aspartic acid, that is distinct from the enzyme active site. Occupation of this site by the substrate, or by various substrate analogues, eliminates both the nonlinearity and the time lag. The D isomer of aspartic acid, which does not bind at the active site, can bind at this newly identified activator site.     

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.     

Mechanism of reaction of 3-hydroxyanthranilic acid with molecular oxygen

The autoxidation of the tryptophan metabolite, 3-hydroxyanthranilic acid, at pH 7 gives rise to a p-quinone dimer and cinnabarinic acid. A novel dimer formed by radical-radical coupling of 3-hydroxyanthranilic acid is also produced. Labelling studies have shown that the C-2 oxygen in the p-quinone dimer is derived from molecular oxygen. A product versus time study of this reaction has revealed that, in the absence of catalase, cinnabarinic acid is formed but undergoes decomposition by hydrogen peroxide. At pH 7, in the presence of catalase, both the p-quinone dimer and cinnabarinic acid are formed at approximately the same rate and this rate of formation increases with increasing pH. Inclusion of superoxide dismutase was found to increase the rate of formation of cinnabarinic acid, suggesting that superoxide ions may also cause decomposition of cinnabarinic acid. This was confirmed by treating cinnabarinic acid with superoxide. A mechanism involving a common anthranilyl radical intermediate is proposed to account for the formation of the different oxidation products.