Environ. Sci. Technol. 2002, 36, 4197-4198
Comment on “Peroxidase-Catalyzed Oxidative Coupling of Phenols in the Presence of Geosorbents: Rates of Non-extractable Product Formation” Huang et al. (1) have reported what may be the first quantitative evaluation of peroxidase inactivation in the presence of soil and demonstrated that soil can decrease the rate of inactivation. The purpose of this letter is to argue that, although eq 6 in the original paper may be appropriate in describing the rate of loss of enzyme activity, the basis from which it was derived is fundamentally unsound. Equation 6 was derived from a proposed scheme presented as Figure 3 in the original paper, which illustrated the formation of a phenoxyl radical via the enzyme-catalyzed oxidation of phenol and subsequent reactions of the phenoxyl radical. One of these reactions is “reverse electron transfer”, or back-conversion of the phenoxyl radical to the parent phenol, which is the focus of my criticism of the overall reaction scheme. The radical formation and reverse electron-transfer reactions are reproduced below: E(kE, KM)
AH2 y\ z AH• reverse electron transfer, k r
(a)
The problem with this coupled “reaction” is that it mixes a composite reaction (the enzyme-catalyzed forward reaction) with an elementary reaction (the reverse reaction). The forward reaction is composite because it lumps the three steps known to occur in the catalytic cycle of peroxidases: the two-electron oxidation of the native peroxidase by hydrogen peroxide followed by two successive one-electron oxidations of a phenol to two phenoxyl radicals. To see the difficulty in coupling the composite forward reaction with the reverse reaction, it is helpful to write out the full reaction stoichiometry: EkE
H2O2 + 2AH2 y\ z 2AH• + 2H2O k r
(b)
The authors cite Taraban et al. (ref 33 in the original paper) as providing experimental evidence for the reverse electrontransfer reaction. Taraban et al. observed, however, a reversible reaction between the phenoxyl radical and one of the oxidized forms of the enzyme within its catalytic cycle. In contrast, the reverse reaction in eq b suggests that phenoxyl radicals will react with water to form hydrogen peroxide (since the enzyme is not changing oxidation states in the composite forward reaction, the enzyme cannot change oxidation states in the reverse reaction either if there is to be a conservation of electrons). This is not the reaction that Taraban et al. had in mind in their work. It therefore would only be appropriate to consider the reverse electron-transfer reaction within the framework of a complete mechanistic treatment of the catalytic cycle in developing an overall equation for the forward reaction. If the authors were to do so, they would find (as with any other enzyme mechanism in which reversible reactions occur) that the Michaelis-Menten equation would still be relevant to the composite forward * Corresponding author phone (919)966-1481; fax: (919)966-7911; e-mail:
[email protected]. 10.1021/es020671s CCC: $22.00 Published on Web 08/31/2002
2002 American Chemical Society
reaction and that the net forward reaction in which phenoxyl radicals form could still be written as in eq a or eq b. The authors claim that reverse electron transfer is likely to be the predominant reaction of the phenoxyl radicals, with a rate presented as eq 3 in the original paper:
rr ) - kr[AH•]
(3)
Equation 3 was used in turn to derive eq 6 to describe the rate of enzyme inactivation:
d[E] ) k′in[E]2 dt
(6)
Data presented in the original Figure 4 are simulated well by eq 6, for experiments conducted both in the presence and absence of soil. Besides the reverse electron-transfer reaction, however, if any reaction of the phenoxyl radicals with something other than another radical is significant, its rate will be of the same form as eq 3 and thus eq 6 would still be valid. Other than reacting with the enzyme itself in the inactivation reaction, in the absence of soil phenoxyl radicals could react with the parent phenol or, more likely, the oligomers that form immediately from radical coupling reactions. Such secondary reactions of radical coupling products, either with the enzyme or with radical species, are not taken into account by the authors; in fact, they are not usually considered in kinetic evaluations of peroxidasecatalyzed reactions. Nevertheless, the fact that the molar stoichiometry of phenol consumption to peroxide consumption approaches 1:1 in peroxidase systems (2) suggests that such secondary reactions are significant. The dimeric coupling products 2,2′-biphenol and 4,4′-biphenol are known to be substrates for horseradish peroxidase (3-5) and have KM values at least 2 orders of magnitude below that of phenol (3). In the presence of soil, cross-coupling reactions of phenoxyl radicals with soil organic matter (SOM) will occur at rates that can also be described by eq 3 (i.e., first-order in phenoxyl radical concentration). Since the analytical procedures were not designed to identify the products as anything but “nonextractable products”, it is not possible to determine the significance of cross-coupling reactions relative to other reactions of phenoxyl radicals. This information is important, however, because cross-coupling reactions will compete with other reactions of the phenoxyl radicals, including the enzyme inactivation reaction. Such competition could lead to the reduced rate of enzyme inactivation observed in the presence of soil. The authors suggested another plausible mechanism for the effect of soil on enzyme inactivation, in which the SOM physically protects sites on the enzyme that are susceptible to attack by phenoxyl radicals; given the empirical nature of the data, it is not possible to distinguish between these mechanisms. However, by invoking the reverse electron-transfer reaction again in the context of the effect of SOM on enzyme inactivation (“the recombination rate constant, kr, should not be affected by the presence of geosorbents”), the authors have virtually dismissed any alternative explanation for their data. While they allude to the possibility that the cross-coupling reactions could reduce the susceptibility of the enzyme to inactivation by the phenoxyl radicals, they do not pursue a quantitative evaluation of this possibility. Overall, the work presented in the original paper led to empirical, but quantifiable, observations. These observations VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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are important. The detailed mechanistic interpretations of the experimental observations are not, however, supportable by the available data. The observations can be described well with rate equations that could be derived from specific reaction mechanisms if there were sufficient data to support those mechanisms; in the absence of such data, the rate equations themselves are empirical. Considering the empirical nature of this study, mechanistic interpretations of the effect of SOM on enzyme inactivation must await more detailed experimental analysis.
Literature Cited (1) Huang, Q.; Selig, H.; Weber, W. J., Jr. Environ. Sci. Technol. 2002, 36, 596-602.
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(2) Caza, N.; Bewtra, J. K.; Biswas, N.; Taylor, K. E. Water Res. 1999, 33, 3012-3018. (3) Danner, D. J.; Brignac Jr., P. J.; Arceneaux, D.; Patel, V. Arch. Biochem. Biophys. 1973, 156, 759-763. (4) Sawahata, T.; Neal, R. A. Biochem. Biophys. Res. Comm. 1982, 109, 988-994. (5) Subrahmanyam, V. V.; O’Brien, P. J. Xenobiotica 1985, 15, 873885.
Michael D. Aitken* Department of Environmental Sciences and Engineering School of Public Health University of North Carolina Chapel Hill, North Carolina 27599-7431 ES020671S
