A computational approach to optimising laccase-mediated polyethylene oxidation through carbohydrate-binding module fusion.

Plastic pollution is a major global concern to the health and wellbeing of all terrestrial and marine life. However, no sustainable method for waste management is currently viable. This study addresses the optimisation of microbial enzymatic polyethylene oxidation through rational engineering of laccases with carbohydrate-binding module (CBM) domains. An explorative bioinformatic approach was taken for high-throughput screening of candidate laccases and CBM domains, representing an exemplar workflow for future engineering research. Molecular docking simulated polyethylene binding whilst a deep-learning algorithm predicted catalytic activity. Protein properties were examined to interpret the mechanisms behind laccase-polyethylene binding. The incorporation of flexible GGGGS(x3) hinges were found to improve putative polyethylene binding of laccases. Whilst CBM1 family domains were predicted to bind polyethylene, they were suggested to detriment laccase-polyethylene associations. In contrast, CBM2 domains reported improved polyethylene binding and may thus optimise laccase oxidation. Interactions between CBM domains, linkers, and polyethylene hydrocarbons were heavily reliant on hydrophobicity. Preliminary polyethylene oxidation is considered a necessity for consequent microbial uptake and assimilation. However, slow oxidation and depolymerisation rates inhibit the large-scale industrial implementation of bioremediation within waste management systems. The optimised polyethylene oxidation of CBM2-engineered laccases represents a significant advancement towards a sustainable method of complete plastic breakdown. Results of this study offer a rapid, accessible workflow for further research into exoenzyme optimisation whilst elucidating mechanisms behind the laccase-polyethylene interaction.

Substituting meat for mycoprotein reduces genotoxicity and increases the abundance of beneficial microbes in the gut: Mycomeat, a randomised crossover control trial.

PURPOSE: The high-meat, low-fibre Western diet is strongly associated with colorectal cancer risk. Mycoprotein, produced from Fusarium venanatum, has been sold as a high-fibre alternative to meat for decades. Hitherto, the effects of mycoprotein in the human bowel have not been well considered. Here, we explored the effects of replacing a high red and processed meat intake with mycoprotein on markers of intestinal genotoxicity and gut health. METHODS: Mycomeat (clinicaltrials.gov NCT03944421) was an investigator-blind, randomised, crossover dietary intervention trial. Twenty healthy male adults were randomised to consume 240 g day-1 red and processed meat for 2 weeks, with crossover to 2 weeks 240 g day-1 mycoprotein, separated by a 4-week washout period. Primary end points were faecal genotoxicity and genotoxins, while secondary end points comprised changes in gut microbiome composition and activity. RESULTS: The meat diet increased faecal genotoxicity and nitroso compound excretion, whereas the weight-matched consumption of mycoprotein decreased faecal genotoxicity and nitroso compounds. In addition, meat intake increased the abundance of Oscillobacter and Alistipes, whereas mycoprotein consumption increased Lactobacilli, Roseburia and Akkermansia, as well as the excretion of short chain fatty acids. CONCLUSION: Replacing red and processed meat with the Fusarium-based meat alternative, mycoprotein, significantly reduces faecal genotoxicity and genotoxin excretion and increases the abundance of microbial genera with putative health benefits in the gut. This work demonstrates that mycoprotein may be a beneficial alternative to meat within the context of gut health and colorectal cancer prevention.

The nutritional impact of replacing dietary meat with meat alternatives in the UK: a modelling analysis using nationally representative data.

Dietary patterns high in meat compromise both planetary and human health. Meat alternatives may help to facilitate meat reduction; however, the nutritional implications of displacing meat with meat alternatives does not appear to have been evaluated. Here, the ninth cycle of the National Diet and Nutrition Survey was used as the basis of models to assess the effect of meat substitution on nutritional intake. We implemented three models; model 1 replaced 25 %, 50 %, 75 % or 100 % of the current meat intake with a weighted mean of meat alternatives within the UK market. Model 2 compared different ingredient categories of meat alternative; vegetable, mycoprotein, a combination of bean and pea, tofu, nut and soya. Model 3 compared fortified v. unfortified meat alternatives. The models elicited significant shifts in nutrients. Overall, carbohydrate, fibre, sugars and Na increased, whereas reductions were found for protein, total and saturated fat, Fe and B12. Greatest effects were seen for vegetable-based (+24·63g/d carbohydrates), mycoprotein-based (-6·12g/d total fat), nut-based (-19·79g/d protein, +10·23g/d fibre; -4·80g/d saturated fat, +7·44g/d sugars), soya-based (+495·98mg/d Na) and tofu-based (+7·63mg/d Fe, -2·02µg/d B12). Our results suggest that meat alternatives can be a healthful replacement for meat if chosen correctly. Consumers should choose meat alternatives low in Na and sugar, high in fibre, protein and with high micronutrient density, to avoid compromising nutritional intake if reducing meat intake. Manufacturers and policy makers should consider fortification of meat alternatives with nutrients such as Fe and B12 and focus on reducing Na and sugar content.

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition.

Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration [I]0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoiding making interpolations between different values of iD. The analytical expression of the IC50, for the different types of inhibition, are related to the apparent inhibition constant (KIapp). Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC50, the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC50 value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in iD and IC50 with substrate concentration. The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition (iD does not depend on [D]0) and the rest. In the case of competitive inhibition, this dependence of iD on [D]0 leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of KI1app is carried out from the IC50 expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of iD vs. n, with n=[D]0/KmD, allows us to distinguish between them. A hyperbolic iD vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of KI2app is immediate from the IC50 value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), KI1app and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), KI2app are obtained from the dependence of iD vs. n, and the results obtained must comply with the IC50 value.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Study of tyrosine and dopa enantiomers as tyrosinase substrates initiating l- and d-melanogenesis pathways.

Tyrosinase starts melanogenesis and determines its course, catalyzing the oxidation by molecular oxygen of tyrosine to dopa, and that of dopa to dopaquinone. Then, nonenzymatic coupling reactions lead to dopachrome, which evolves toward melanin. Recently, it has been reported that d-tyrosine acts as tyrosinase inhibitor and depigmenting agent. The action of tyrosinase on the enantiomers of tyrosine (l-tyrosine and d-tyrosine) and dopa (l-dopa and d-dopa) was studied for the first time focusing on quantitative transient phase kinetics. Post-steady-state transient phase studies revealed that l-dopachrome is formed more rapidly than d-dopachrome. This is due to the lower values of Michaelis constants for l-enantiomers than for d-enantiomers, although the maximum rates are equal for both enantiomers. A deeper analysis of the inter-steady-state transient phase of monophenols demonstrated that the enantiomer d-tyrosine causes a longer lag period and a lower steady-state rate, than l-tyrosine at the same concentration. Therefore, d-melanogenesis from d-tyrosine occurs more slowly than does l-melanogenesis from l-tyrosine, which suggests the apparent inhibition of melanin biosynthesis by d-tyrosine. As conclusion, d-tyrosine acts as a real substrate of tyrosinase, with low catalytic efficiency and, therefore, delays the formation of d-melanin.

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the d-galacturonic acid-α-1,2-l-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second d-galacturonic acid side chain (linked ß-1,3 to l-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/ß)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting d-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.

Unusual active site location and catalytic apparatus in a glycoside hydrolase family.

The human gut microbiota use complex carbohydrates as major nutrients. The requirement for an efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that these bacteria represent a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis, we focused on enzymes that hydrolyze rhamnosidic bonds, as cleavage of these linkages is chemically challenging and there is a paucity of information on l-rhamnosidases. Here we screened the activity of enzymes derived from the human gut microbiota bacterium Bacteroides thetaiotaomicron, which are up-regulated in response to rhamnose-containing glycans. We identified an α-l-rhamnosidase, BT3686, which is the founding member of a glycoside hydrolase (GH) family, GH145. In contrast to other rhamnosidases, BT3686 cleaved l-Rha-α1,4-d-GlcA linkages through a retaining double-displacement mechanism. The crystal structure of BT3686 showed that the enzyme displayed a type A seven-bladed ß-propeller fold. Mutagenesis and crystallographic studies, including the structure of BT3686 in complex with the reaction product GlcA, revealed a location for the active site among ß-propeller enzymes cited on the posterior surface of the rhamnosidase. In contrast to the vast majority of GH, the catalytic apparatus of BT3686 does not comprise a pair of carboxylic acid residues but, uniquely, a single histidine functions as the only discernable catalytic amino acid. Intriguingly, the histidine, His48, is not invariant in GH145; however, when engineered into structural homologs lacking the imidazole residue, α-l-rhamnosidase activity was established. The potential contribution of His48 to the catalytic activity of BT3686 is discussed.

The human gut microbe Bacteroides thetaiotaomicron encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29.

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.

An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.

The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of Bacteroides thetaiotaomicron that were up-regulated by arabinogalactan proteins or AGPs. Although AGPs are ubiquitous in plants, there is a paucity of information on their detailed structure, the function of these glycans in planta, and the mechanisms by which they are depolymerized in microbial ecosystems. Here we have discovered a new polysaccharide lyase family that is specific for the l-rhamnose-α1,4-d-glucuronic acid linkage that caps the side chains of complex AGPs. The reaction product generated by the lyase, Δ4,5-unsaturated uronic acid, is removed from AGP by a glycoside hydrolase located in family GH105, producing the final product 4-deoxy-ß-l-threo-hex-4-enepyranosyl-uronic acid. The crystal structure of a member of the novel lyase family revealed a catalytic domain that displays an (α/α)6 barrel-fold. In the center of the barrel is a deep pocket, which, based on mutagenesis data and amino acid conservation, comprises the active site of the lyase. A tyrosine is the proposed catalytic base in the ß-elimination reaction. This study illustrates how highly complex glycans can be used as a scaffold to discover new enzyme families within microbial ecosystems where carbohydrate metabolism is a major evolutionary driver.

Catalysis and inactivation of tyrosinase in its action on hydroxyhydroquinone.

Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mechanism is proposed, in which HHQ is considered as a monophenol or as an o-diphenol, depending on the part of the molecule that interacts with the enzyme. The kinetic parameters obtained from an analysis of the measurements of the initial steady state rate of 2-hydroxy p-benzoquinone formation were kcatapp=229.0±7.7 s(-1) and KMapp,HHQ=0.40±0.05 mM. Furthermore, the action of tyrosinase on HHQ led to the enzyme's inactivation through a suicide inactivation mechanism. This suicide inactivation process was characterized kinetically by λmaxapp (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of λmaxapp and r were (8.2±0.1)×10(-3) s(-1) and 35,740±2,548, respectively.

Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study.

Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC-MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed.

Indirect inactivation of tyrosinase in its action on 4-tert-butylphenol.

Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates met and deoxytyrosinase enzymatic forms of tyrosinase. However, the monophenol 4-tert-butylphenol (TBF) protects the enzyme from this inactivation. Under aerobic conditions, the enzyme suffers suicide inactivation when it acts on TBC. We suggest that TBF does not directly cause the suicide inactivation of the enzyme in the hydroxylase activity, but that the o-diphenol, which is necessary for the system to reach the steady state, is responsible for the process. Therefore, monophenols do not induce the suicide inactivation of tyrosinase in its hydroxylase activity, and there is a great difference between the monophenols that give rise to unstable o-quinones such as L-tyrosine, which rapidly accumulate L-dopa in the medium and those like TBF, after oxidation, give rise to a very stable o-quinone.

A review of the principles and biotechnological applications of glycoside hydrolases from extreme environments.

It is apparent that Biocatalysts are shaping the future by providing a more sustainable approach to established chemical processes. Industrial processes rely heavily on the use of toxic compounds and high energy or pH reactions, factors that both contributes to the worsening climate crisis. Enzymes found in bacterial systems and other microorganisms, from the glaciers of the Arctic to the sandy deserts of Abu Dhabi, provide key tools and understanding as to how we can progress in the biotechnology sector. These extremophilic bacteria harness the adaptive enzymes capable of withstanding harsh reaction conditions in terms of stability and reactivity. Carbohydrate-active enzymes, including glycoside hydrolases or carbohydrate esterases, are extremely beneficial for the presence and future of biocatalysis. Their involvement in the industry spans from laundry detergents to paper and pulp treatment by degrading oligo/polysaccharides into their monomeric products in almost all detrimental environments. This includes exceedingly high temperatures, pHs or even in the absence of water. In this review, we discuss the structure and function of different glycoside hydrolases from extremophiles, and how they can be applied to industrial-scale reactions to replace the use of harsh chemicals, reduce waste, or decrease energy consumption.

Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum.

Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.

Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase.

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamineso-aminophenols>aromatic o-diamines.

Deuterium isotope effect on the suicide inactivation of tyrosinase in its action on o-diphenols.

A solvent deuterium isotope effect on the inactivation suicide of tyrosinase in its action on o-diphenols (catechol, 4-methylcatechol, and 4-tert-butylcatechol) was observed. This isotope effect, observed during kinetic studies in the transition phase, was higher than that described previously in the steady state, indicating that there is an additional slow step in the suicide inactivation mechanism, which we believe to be responsible for the inactivation. In a proton inventory study of oxidation of o-diphenols, the representation of λmax(D,fn)/λmax(D,f0) versus n (atom fractions of deuterium), where λmax(D,fn) is the maximum apparent inactivation constant for a molar fraction of deuterium (n) and λmax(D,f0) is the corresponding kinetic parameter in a water solution, was linear for all substrates. This suggests that only one of the protons transferred from the two hydroxyl groups of the substrate, which are oxidized in one turnover, is responsible for the isotope effects. We propose that this proton could be the proton transferred from the hydroxyl group of C-2 to the hydroperoxide of the oxytyrosinase form (Eox ) and that it probably causes enzyme inactivation through the reduction of the Cu(2+) A to Cu(0) and its subsequent release from the active site.