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.

Sand gapers’ breath: Respiration of Mya arenaria (L. 1758) and its contribution to total oxygen utilization in sediments

2019 ◽  
Vol 143 ◽  
pp. 101-110 ◽  
Author(s):  
Hanna Schade ◽  
Nikolas Arneth ◽  
Martin Powilleit ◽  
Stefan Forster
Keyword(s):  
Mya Arenaria ◽  
Oxygen Utilization ◽  
Total Oxygen

scholarly journals The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 951-957 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Lactate Dehydrogenase ◽  
Dissociation Constant ◽  
Active Site ◽  
Adduct Formation ◽  
Rabbit Muscle ◽  
Muscle Lactate

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 × 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195–1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.

Pumping Rates, Water Pressures, and Oxygen Use in Eight Species of Marine Bivalve Molluscs from British Columbia

1990 ◽  
Vol 47 (7) ◽  
pp. 1302-1306 ◽  
Author(s):  
F. R. Bernard ◽  
D. J. Noakes
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Cost ◽  
Small Samples ◽  
Maximum Pressure ◽  
Marine Bivalve ◽  
Bivalve Molluscs ◽  
Oxygen Utilization ◽  
Total Oxygen ◽  
Anoxic Environments ◽  
Pumping Rates

Pumping characteristics and oxygen utilization for Solemya reidi, Yoldia thraciaeformis, Chlamys hastata, Mytilus edulis, Crassostrea gigas, Clinocardium nuttallii, Saxidomus giganteus, and Mya truncata were studied. Pressures were recorded using a Yale A79 spinal tap needle inserted in the pallial cavity or siphonal aperture. Pumping volumes were determined through particulate analysis using a Model B Coulter counter and oxygen consumption by standard Winkler titration modified for small samples. The species chosen were selected to represent progressive increases in gill complexity and siphon length. The minimum pressure differential across the gill (20 Pa) was observed for Y. thraciaeformis while the maximum pressure gradient (600 Pa) was recorded for M. truncata. Pumping rates varied from 1.41 L∙h−1∙g−1 (Y. thraciaeformis) to 4.71 L∙h−1∙g−1 (C. hastata) and species with siphons tended to pump at lower rates. The deeply burrowing M. truncata consumed 3.5 times as much oxygen (0.63 mL O2∙h−1∙g−1) as S. reidi (0.18 mL O2∙h−1∙g−1) which inhabits anoxic environments. For all species, the metabolic cost of pumping was less than 1% of the total oxygen uptake.

scholarly journals Tyrosinase Inhibition Kinetics of Anisic Acid

2003 ◽  
Vol 58 (9-10) ◽  
pp. 713-718 ◽  
Author(s):  
Isao Kubo ◽  
Qing-Xi Chen ◽  
Ken-Ichi Nihei ◽  
José S. Calderón ◽  
Carlos L. Céspedes
Keyword(s):  
Lag Phase ◽  
Tyrosinase Inhibitor ◽  
Inhibition Kinetics ◽  
Tyrosinase Inhibition ◽  
Monophenolase Activity ◽  
Anisic Acid ◽  
Steady State Activity ◽  
Noncompetitive Inhibitor ◽  
Kinetics Of ◽  
Methoxybenzoic Acid

AbstractAnisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from aniseed, a common food spice. It inhibited the oxidation of l-3,4-dihydroxyphenylalanine (ʟ- DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mᴍ. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant KI was obtained as 0.603 mᴍ. Anisic acid also inhibited the hydroxylation of ʟ-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.

scholarly journals Synthesis of Linoleic Acid 13-Hydroperoxides from Safflower Oil Utilizing Lipoxygenase in a Coupled Enzyme System with In-Situ Oxygen Generation

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1119
Author(s):  
Valentin Gala Marti ◽  
Anna Coenen ◽  
Ulrich Schörken
Keyword(s):  
Linoleic Acid ◽  
Candida Rugosa ◽  
Reaction System ◽  
Lag Phase ◽  
Safflower Oil ◽  
Reaction Conditions ◽  
Free System ◽  
Enzyme Cascade ◽  
Oil Hydrolysis

Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.

scholarly journals Spectroscopic characterization of thiol adducts formed in the reaction of 4-methylcatechol with DPPH in the presence of N-acetylcysteine

2018 ◽  
Vol 9 (4) ◽  
pp. 386-393
Author(s):  
Masaki Ichitani ◽  
Hisako Okumura ◽  
Yugo Nakashima ◽  
Hitoshi Kinugasa ◽  
Mitsunori Honda ◽  
...  
Keyword(s):  
Radical Scavenging Activity ◽  
Direct Reduction ◽  
Radical Scavenging ◽  
Spectroscopic Characterization ◽  
Major Product ◽  
Preparative Hplc ◽  
Thiol Compounds ◽  
Activity Enhancement ◽  
Dpph Radical Scavenging

Nucleophiles such as thiol compounds have enhancing effects on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of polyphenols. Several authors have suggested that regeneration of the catechol structure from o-quinone plays a key role in enhanced radical scavenging activity. We therefore explored the reaction of 4-methyl catechol (MC) with DPPH in the presence of N-acetylcysteine (NACys) to clarify the mechanism underlying activity enhancement. Four types of NACys adducts were isolated and purified by preparative HPLC after the reactions reached equilibrium and their structures were characterized spectroscopically using UV-Vis absorption, NMR, and LC-MS. Oxidation of MC using a periodate resin and subsequent reaction with NACys were also studied. LC-MS analyses revealed that a mono-NACys adduct is produced as the major product in the reaction of MC quinone with NACys, and direct reduction by NACys occurs in reactions with NACys MC quinones.

scholarly journals Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

2018 ◽  
Author(s):  
PV Dayal ◽  
HR Ellis
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Lag Phase ◽  
Monooxygenase System ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Loop Region ◽  
Alkanesulfonate Monooxygenase ◽  
Α Helix

AbstractBacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved α-helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of α-helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the α-helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The α-helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.

Regulation of NADP-Malate Dehydrogenase in C4 Plants: Activity and Properties of Maize Thioredoxin M and the Significance of Non-Active Site Thiol Groups

1995 ◽  
Vol 22 (4) ◽  
pp. 577 ◽  
Author(s):  
JE Lunn ◽  
A Agostino ◽  
MD Hatch
Keyword(s):  
Malate Dehydrogenase ◽  
Active Site ◽  
Covalent Binding ◽  
Molar Ratio ◽  
Stable Complex ◽  
Thiol Groups ◽  
Thiol Compounds ◽  
Maize Leaf

Some unusual properties of purified maize leaf thioredoxin m were attributable to the presence of non-active site thiol groups, Unlike thioredoxins from other sources, maize leaf thioredoxin m was susceptible to inactivation by heating and this was associated with polymerisation of the molecule. Both these effects of heating were prevented or reversed by adding thiol compounds such as dithiothreitol. We concluded that, on heating, the free SH groups in the oxidised thioredoxin m molecule react with disulfide groups of other molecules to form polymeric complexes linked by disulfide bonds. We also observed the formation of a stable complex between the oxidised forms of thioredoxin m and NADP-malate dehydrogenase (NADP-MDH) under certain conditions. Evidence that in maize chloroplasts thioredoxin m and NADP-MDH occur in a 1: 1 molar ratio suggested that they may exist as a complex in vivo. However, ratios of thioredoxin rn to NADP-MDH varied widely in other species but always with thioredoxin m in excess. Furthermore, the complex we observed in vitro was shown to be the result of intermolecular disulfide bond formation and apparently occurred only with a non-physiological form of oxidised thioredoxin m. We could not demonstrate any non-covalent binding between thioredoxin m and NADP-MDH.

Cardiovascular–Respiratory Activity During Recovery from Anesthesia and Surgery in Brook Trout (Salvelinus fontinalis) and Carp (Cyprinus carpio)

1973 ◽  
Vol 30 (11) ◽  
pp. 1705-1712 ◽  
Author(s):  
A. H. Houston ◽  
C. L. Czerwinski ◽  
R. J. Woods
Keyword(s):  
Cyprinus Carpio ◽  
Brook Trout ◽  
Salvelinus Fontinalis ◽  
Lag Phase ◽  
Oxygen Utilization ◽  
Functional Variation ◽  
Carp Cyprinus Carpio ◽  
Recovery From Anesthesia ◽  
Rate Ratios

Brook trout (Salvelinus fontinalis) and carp (Cyprinus carpio) were subjected to handling, anesthesia, and extensive experimental pretreatment, including the emplacement of electrocardiograph electrodes and of buccal, cleithral, dorsal aortic, and urinary bladder catheters. Determination of oxygen consumption, cardiac and ventilatory rates, and rate ratios, ventilatory flow, ventilatory stroke volume, and gross oxygen utilization were subsequently carried out at intervals over a 26-hr postpreparation period. Four general patterns of functional variation were observed during recovery. In the most common pattern, activity changed steadily to a final value. Other patterns were characterized by an initial lag phase or intermediate maximum, minimum, or plateau values prior to apparent stabilization. Brook trout required longer than carp for recovery, and differed from carp in the relationship between ventilation and oxygen utilization during recovery. Provision for recovery periods of not less than 24 hr is recommended for studies involving preexperimental preparations of the type studied.

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