scholarly journals Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate

1998 ◽  
Vol 333 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Christopher J. COOKSEY ◽  
Peter J. GARRATT ◽  
Edward J. LAND ◽  
Christopher A. RAMSDEN ◽  
Patrick A. RILEY
Keyword(s):  
Rate Constant ◽  
Pulse Radiolysis ◽  
Phenol Oxidation ◽  
Activation Mechanism ◽  
Oxygen Stoichiometry ◽  
Oxygen Utilization ◽  
Maximum Acceleration ◽  
Semiquinone Radicals ◽  
Catalysed Oxidation ◽  
Dihydric Phenol

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1·cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol·min-1 and the apparent Km was 374 µM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol·min-1 and Km 309 µM. The quinomethane was found to decay with a rate constant of 2k 38 M-1·s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.

scholarly journals Hydrated electrons induce the formation of interstrand cross-links in DNA modified by cisplatin adducts

2020 ◽  
Vol 61 (3) ◽  
pp. 343-351
Author(s):  
B Behmand ◽  
A M Noronha ◽  
C J Wilds ◽  
J-L Marignier ◽  
M Mostafavi ◽  
...  
Keyword(s):  
Rate Constant ◽  
Gamma Rays ◽  
Gel Electrophoresis ◽  
Pulse Radiolysis ◽  
Interstrand Crosslinks ◽  
Radiosensitizing Effect ◽  
Hydrated Electrons ◽  
Interstrand Cross Links ◽  
Cross Links

Abstract Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M−1 s−1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair.

Conductometric Pulse Radiolysis Study on the Reaction of the Solvated Electron with 5-Bromouracil in Aqueous Solutions at Different pH Values

1974 ◽  
Vol 29 (1-2) ◽  
pp. 86-88b ◽  
Author(s):  
Burkhard O. Wagner ◽  
Herbert Klever ◽  
Dietrich Schulte-Frohlinde
Keyword(s):  
Aqueous Solution ◽  
Hydrogen Atom ◽  
Aqueous Solutions ◽  
Hydroxyl Radical ◽  
Rate Constant ◽  
Pulse Radiolysis ◽  
Solvated Electron ◽  
Ph Values

To study the reaction of the solvated electron with 5-bromouracil an aqueous solution has been examined by conductometric pulse radiolysis at pH values between 4.68 and 8.74. Alcohol was added to scavenge the hydrogen atom and the hydroxyl radical. G(Br—) = (2.64 ± 0.08)/100 eV was found to be independent of the pH. The mobility of the bromouracil mono-anion was measured to be (2.7 ± 0.2) 10-4 cm2 V-1 s-1 at 20°C, and the rate constant of reaction (3b) was determined to be k(H+ BrUr-) = (2.3 ± 0.2) 1010 I mole-1 s-1*.

scholarly journals A computational study of the fluctional behaviour of group 14 substituted ortho-semiquinone radicals

2011 ◽  
Vol 89 (2) ◽  
pp. 235-240 ◽  
Author(s):  
K. U. Ingold ◽  
Gino A. DiLabio
Keyword(s):  
Experimental Data ◽  
Density Functional Theory ◽  
Rate Constant ◽  
Density Functional ◽  
Computational Study ◽  
Group 14 ◽  
Functional Theory ◽  
Semiquinone Radicals ◽  
Experimental Values ◽  
Good Agreement

The dynamics of the 1,4-migration of some O-substituted 3,5-di-tert-butyl-ortho-semiquinone radicals have been calculated by density-functional theory (DFT). There is very good agreement in the rate constant and Arrhenius parameters between these calculations and experimental values for migration of H, D, and the Me3Si group. For the Me3Sn group, the calculations indicate an incredibly fast migration (k293K = 2.0 × 1012 s–1), a result that is consistent with experimental data (k293K > 109 s–1). Other O-substituents examined by DFT and compared with experimental data were H3C and Me2ClSn.

Peptide Free-Radicals: The Reactions of OH Radicals with Glycine Anhydride and its Methyl Derivatives Sarcosine and Alanine Anhydride. A Pulse Radiolysis and Product Study

1989 ◽  
Vol 44 (8) ◽  
pp. 959-974 ◽  
Author(s):  
Oliver J. Mieden ◽  
Clemens von Sonntag
Keyword(s):  
Hydrogen Atom ◽  
Dose Rate ◽  
Rate Constant ◽  
Pulse Radiolysis ◽  
Solute Concentration ◽  
Oh Radicals ◽  
Initial Attack ◽  
Product Analysis ◽  
H Nmr ◽  
Methyl Derivatives

The reactions of radiolytically generated OH radicals and H atoms with the cyclic dipeptides of glycine, alanine and sarcosine in deoxygenated aqueous solutions and the subsequent reactions of the transient peptide radicals were studied in the absence and presence of K3Fe(CN)6 as oxidant by pulse radiolysis and product analysis.Hydroxyl radicals and H atoms react with glycine anhydride and alanine anhydride by abstracting an H atom bound at C-3; there is no evidence for any other site of attack at these two peptides. The resulting radicals have pKa values of 9.8 and 10.6, respectively.In the absence of an oxidant the radicals decay by second order (2k = 7.0×108 dm3 mol-1 s-1 and 2k = 4.4×108 dm3 mol-1 s-1, resp.), the main fraction (94% of the glycine anhydride-derived radicals, 90% of the alanine anhydride-derived radicals) yielding dehydrodimers (G = 0.58 μmol J-1 and 0.56 µmol J-1 (in monomer units), resp.). A small portion however disproportionates via abstraction of a C-6-bound Η atom followed by isomerization to 2,5-dihydroxypyrazines (pKa values of the parent 2,5-dihydroxypyrazine at about 7.9 and 10.1) and subsequent addition of water to 2,5-diketo-3-hydroxypiperazines, thus indicating that the transfer of a carbon-bound hydrogen atom is prefered to the transfer of a nitrogen-bound hydrogen atom.No disproportionation products but three different dehydrodimers (G = 0.36, 0.18 and 0.04 µmol J-1 (in monomer units)) were found after irradiation of sarcosine anhydride. In this case a dose rate and solute concentration dependence of dehydrodimer formation indicates a radical-solute reaction converting part of the N-methyl radicals (21% of ‘initial’ attack) into the C-3-yl radicals. A rate constant of k = 600 ± 50 dm3 mol-1 s-1 was obtained for this reaction by measuring and computing the dehydrodimer yields as a function of dose rate and solute concentration. Thus the observed transient spectrum accounts only for about 79% of the radicals from the ‘initial’ attack at C-3.The rate of oxidation of the glycine anhydride-derived radicals by Fe(CN)63- reflects the pKa of the transient radical. The rate constant for oxidation of the (protonated) radical derived from glycine anhydride is: k = 1.0x 108 dm3 mol-1, the corresponding radical anion is oxidized with k = 3.1 × 108 dm3 mol-1 s-1. No change with pH was observed in the case of the alanine anhydridederived radicals (k = 7.9x 108 dm3 mol-1 s-1). In contrast to the disproportionation, oxidation by Fe(CN)63- leads to the removal of a proton from the heteroatom, a carbocation being the intermediate. The resulting dehydropiperazines rapidly add water to yield the corresponding 2,5-diketo-3-hydroxypiperazines (G = 0.61 μmol J-1 after oxidation of the glycine anhydride-derived radicals, G = 0.58 µmol J-1 after oxidation of the alanine anhydride-derived radicals). The radicals derived from sarcosine anhydride are readily oxidized with k = 4.0×108 dm3 mol-1 s-1, independent of pH.1H and 13C{1H} NMR-spectroscopic and mass-spectroscopic data of the products are given.

Pulse radiolysis: evidence for the reaction of HO2 with NO2 in aqueous solution

1978 ◽  
Vol 56 (14) ◽  
pp. 1961-1964 ◽  
Author(s):  
Harry Callender Sutton ◽  
William Arthur Seddon ◽  
Fred Charles Sopchyshyn
Keyword(s):  
Aqueous Solution ◽  
Formic Acid ◽  
Rate Constant ◽  
Pulse Radiolysis ◽  
First Order ◽  
Data Support ◽  
Radiation Induced ◽  
Three Stages ◽  
Ph Range ◽  
Saturated Solutions

Nitroform is the major radiation induced species observed after the pulse radiolysis of acidic oxygen saturated solutions of tetranitromethane (TNM) and formic acid. It is formed in three stages, of which a major component at pH < 3 is first order with t1/2 = 49 s, independent of [TNM] and pH. Evidence is provided for the reactions[Formula: see text]in which k14 = 0.014 ± 0.002 s−1. The data support similar conclusions reached previously in rapid mixingexperiments (1) from which it was concluded that k3 = 4 × 109 dm3 mol−1 s−1.Analysis of the fastest component of nitroform production over the pH range 1–4 shows that the COOH radical reduces TNM to nitroform with a rate constant about four times greater than that for its reaction with oxygen to produce HO2.

scholarly journals Formation of super-reduced Chromatium high-potential iron–sulphur protein in aqueous solution by pulse radiolysis

1980 ◽  
Vol 189 (3) ◽  
pp. 641-644 ◽  
Author(s):  
J Butler ◽  
A G Sykes ◽  
G V Buxton ◽  
P C Harrington ◽  
R G Wilkins
Keyword(s):  
Aqueous Solution ◽  
Rate Constant ◽  
Rate Constants ◽  
Pulse Radiolysis ◽  
High Potential ◽  
Single Stage ◽  
Process Rate ◽  
Stage Process ◽  
Reduced Forms

Both the oxidized and reduced forms of Hipip (high-potential iron–sulphur protein) are reduced (approx. 30% yields) by eaq.- in a single-stage process, rate constants 1.7 × 10(10) and 1.8 × 10(10) M-1 . s-1 respectively, at 25 degrees C, pH 7.0 (5 mM-phosphate). Super-reduced Hipip, which is formed in the latter case, has a spectrum which closely resembles that of reduced ferredoxin, i.e. Fe4S4 (SR)4(3-) clusters. The spectrum is stable over 2 s periods investigated. Super-reduced Hipip is reoxidized with O2, rate constant 4.8 × 10(6) M-1 . s-1 at 25 degrees C.

scholarly journals Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics

1994 ◽  
Vol 304 (1) ◽  
pp. 155-162 ◽  
Author(s):  
S Naish-Byfield ◽  
C J Cooksey ◽  
P A Riley
Keyword(s):  
Active Site ◽  
Direct Reduction ◽  
Reaction System ◽  
Adduct Formation ◽  
Lag Phase ◽  
Oxygen Utilization ◽  
Total Oxygen ◽  
Thiol Compounds ◽  
Monophenolase Activity ◽  
Dihydric Phenol

The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.

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