Structure, function and operational stability of peroxidases

M.J.H. van Haandel

Research output: Thesisinternal PhD, WU

Abstract

<p>This PhD project was started in 1995 and was supported by the dutch Ministry of Economic Affairs through the programme "IOP Catalysis". The main goal of "IOP catalysis" is to obtain clean and more efficient technologies, to improve the quality of the Dutch fine chemistry. Biocatalysis provides a way to minimize non desirable side products, which can have a negative impact on the environment. The objective of this thesis was to investigate the potential of heme-containing peroxidases as natural biocatalysts for industrially relevant conversions. Peroxidases were chosen since they are able to operate under mild conditions using cheap and clean oxidants (hydrogen peroxide).</p><p>In this work especially the natural production of food flavours was of interest. After initial screening of reactions of potential interest the N-dealkylation of methyl-N-methylanthranilate) (ex citrus leaves) to methylanthranilate (MA), a concord grape flavour was chosen as the mode) reaction in this thesis.</p><p>In order to obtain better insight in the industrial applications of peroxidases, it is important to understand structure, function and operational stability of these catalysts. Therefore, this project was started by investigating the reaction mechanism of peroxidases in more detail ( <strong>chapter 3</strong> ). Horseradish peroxidase (HRP) was used as the model peroxidase, as this enzyme is the best studied enzyme of all peroxidases. Many heme-containing biocatalysts, exert their catalytic action through the initial formation of so-called high-valent- iron-oxo porphyrin intermediates. For HRP the initial intermediate formed has been reported as a high-valent-iron-oxo porphyrinπ-radical cation, called compound I. A strongly hold concept in the field of peroxidase-type of catalysis is the irreversible character of the reaction leading to formation of this compound I. The results of chapter 3, however, indicate that formation of the high-valent-iron-oxo porphyrin intermediate for various heme-containing catalysts, including HRP, might be reversible. This reversible compound I formation results in heme-catalysed exchange of the oxygens of H <sub>2</sub> O <sub>2</sub> with those of H <sub>2</sub> O. The existence of this heme-catalysed oxygen exchange followed from the observation that upon incubation of <sup>18</sup> O labeled H <sub>2</sub><sup>18</sup> O <sub>2</sub> with heme-containing biocatalysts, significant loss of the <sup>18</sup> O label from the H <sub>2</sub><sup>18</sup> O <sub>2</sub> was observed, accompanied by the formation of unlabeled H <sub>2</sub> O <sub>2</sub> . Thus, for the heme biocatalysts studied, exchange of the oxygen of their high-valent-iron-oxo intermediate with that of water occurs rapidly. This observation implied the need for an update of the kinetic model for peroxidases. Revaluation and extension of the previous kinetic model for HRP showed the necessity to include several additional reaction steps, taking both reversible compound I formation and the formation of enzyme-substrate complexes into account As a consequence reactions with HRP are saturable, implying that V <sub>max</sub> and k <sub>cat</sub> can be measured. This provides possibilities for investigation of structure-activity relationships.</p><p>Investigation of quantitative structure-activity relationships (QSARs) is a way to obtain more insight in the influence of the structure of a substrate on its conversion by an enzyme of interest. Moreover, QSARs could be of interest for industrial applications. With QSARs the outcomes of conversions may be predicted by simply calculating chemical parameters of structurally related substrates. In this way QSARs could be helpful in facilitating screening procedures for biocatalytic productions, saving time and money.</p><p>In <strong>chapter 4</strong> predictive computer calculation-based QSARs were defined by comparing second order rate constants for the oxidation of a series of model compounds by HRP compound II to computer calculated chemical parameters characteristic for this reaction step. The model compounds studied were a series of structurally related phenols. For the calculation of the chemical parameters characteristic for the reaction step two approaches were used. In the first approach a frontier orbital parameter of the substrate was calculated being the ionisation potential (i.e. minus the energy of their highest occupied molecular orbital (E(HOMO)). In the second approach the relative heat of formation (ΔΔHF) was calculated for the process of one electron abstraction as well as for H·-abstraction from the phenol derivatives. Assuming a reaction of the phenotic substrates in their non- dissociated, uncharged forms, clear correlations were obtained between the natural logarithm of the second order rate constants (ln k <sub>app</sub> and ln k <sub>2</sub> respectively ) for their oxidation by compound II and their calculated parameters.</p><p>The computer calculation-based QSARs thus obtained for the oxidation of the various phenol derivatives by compound II from HRP indicate the validity of the approaches investigated, i.e. the frontier orbital approach but also the approach io which the process is described by calculated relative heats of formation. The results also indicated that outcomes from computer calculations on related phenol derivatives can be reliably compared to one another. Since both mechanisms considered, i.e. initial electron abstraction versus initial H·-abstraction provided clear molecular orbital QSARs the results could not be used to discriminate between these two possible mechanisms for phenol oxidation by HRP compound II. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known to be the rate-limiting step in the overall catalysis by HRP, the QSARs described in chapter 4 may have implications for the differences in the overall rate of oxidation of the phenol derivatives by HRP. As a matter of fact similar QSARs should be obtained when the overall rate of oxidation of the respective compounds by HRP is determined.</p><p>As mentioned above, reactions with HRP are saturable, implying that V <sub>max</sub> and k <sub>cat</sub> can be measured. Thus, in <strong>chapter 5</strong> the overall conversion of phenols by HRP was investigated, resulting in overall k <sub>cat</sub> values. These saturating overall k <sub>cat</sub> values indeed correlated quantitatively with calculated ionisation potentials of the substrates. The observation that the rates and QSARs obtained for the overall rate of conversion in chapter 5 are similar to those described for the rate limiting reaction step in chapter 4, corroborates that phenol oxidation by compound II is the rate limiting step in the reaction cycle of HRP, but even more important, it also illustrates that QSARs for oxidation of substrates by compound II can be used for prediction of the overall rate of oxidation of phenol derivatives by HRP and vice versa. Moreover, QSARs for overall k <sub>cat</sub> , instead of for individual rate constants, eliminate the need for extensive rapid kinetic analysis to make predictions for HRP-based substrate conversions.</p><p>Saturating overall k <sub>cat</sub> values for HRP catalysed conversion of a second series of substrates, i.e. a series of substituted anilines, described in chapter 5, also correlated quantitatively with calculated ionisation potentials of the substrates. However, in the QSAR plots, the correlations for the anilines were shifted to lower k <sub>cat</sub> values at similar ionisation potentials as compared to those for the phenols. To investigate whether differences in orientation of phenols and anilines within the active site of HRP may be a factor underlying the higher reactivity of the phenols than expected on the basis of their ionisation potential, <sup>1</sup> H NMR <em>T <sub>1</sub></em> relaxation studies were performed, using 3-methylphenol aod 3-methylaniline as the model substrates. The <sup>1</sup> H NMR <em>T <sub>1</sub></em> relaxation studies revealed consistently smaller average distances of the phenol than of the aniline protons to the paramagnetic Fe <sup>3+</sup> centre in HRP, may be resulting in differences io the electron transfer process from the aromatic donor substrate to the Compound II of HRP. A shorter distance between the phenol and the heme than between the aniline and the heme may be a factor contributing to the faster rate of electron transfer with phenol as compared to aniline substrates. However, the actual differences in orientation seem small when the difference in oxidation rate at similar calculated ionisation potential phenols or anilines is considered. Since (partial) deprotonation will largely influence, i.e. decrease the ionisation potential of the aromatic substrate, the relatively higher oxidation rates of phenols may be related to their larger extent of deprotonation upon binding to the substrate pocket of HRP, resulting in lower ionisation potentials than actually expected on the basis of calculations on their non-ionised form. Based on the <sup>1</sup> H NMR <em>T <sub>1</sub></em> relaxation data of chapter 4 and literature data of Hendriksen et al. [<a href="#ref">1</a>], we put forward the hypothesis that the differential substrate behaviour of phenols and anilines may be due to subtle differences in their binding to the active site substrate pocket of HRP, resulting in i) closer proximity to the heme and ii) larger extent of deprotonation for the phenols than for the aniline substrates. An important conclusion following from the results of chapter 5 is that for each type of reaction and substrate, different QSARs have to be obtained. It is also clear that further investigations are nessecary for succesful application of QSARs to industrial prediction of biocatalysis.</p><p>Heme-based peroxidases are enzymes with a broad substrate specificity and are capable of catalysing a variety of reactions. However, operational (in)stability limits the use of peroxidases in industrial processes. <strong>Chapter 6</strong> describes the possibillities and limitations for using commercially available SP preparations (delivered by QUEST) and other peroxidases (like HRP and microperoxidase-8 (MPS)) for the production of the food flavour methylanthranilate (MA) from methyl-N-methylanthranilate (MNMA). Because W from citrus leaves is a relatively cheap source and MA is more expensive than MNMA, the investigated reaction provides an industrially relevant route for the natural production of an important topnote flavour in concord grape.</p><p>The tested soybean preparations varied widely with respect to their heme-content and, as a result, their activity for this reaction. Furthermore, the operational stability of purified soybean peroxidase (SP) was at least 25-fold lower than that of HRP and only 5-fold higher than that of MP8, a small peroxidase with a polypeptide chain of only eight amino acids covalently linked to the protoporphyrin IX cofactor. Therefore, the results with SP indicate that the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stabifity, and that the inactivation mechanism could be intramolecular. In order to find an explanation for the low operational stability of SP and MP8, it would therefore be interesting to investigate, for example, the shielding of the meso-positions of the heme cofactors of the different peroxidases.</p><p>All tested peroxidase preparations were able to catalyse the requested N-dealkylation. However, SP proved to be a very efficient biocatalyst for the production of MA with high yield and purity, in spite of its relatively low operational stability. This potential of SP to catalyse the N-demethylation of MNMA to MA more efficiently than HRP and MP8, was especially observed at high temperature and low pH values at which SP appeared to be optimally active. Unfortunately, the prices for MA had dropped by the time this efficient SP catalysed production of MA was developed. As a result the now defined way to produce MA using a peroxidase biocatalyst was no longer of commercial interest for the industrial IOP partner. Nevertheless, the results in chapter 6 clearly define the answer to the initial industrial aim of the preseot IOP project.</p><h3>Biotechnological applications</h3><p>Peroxidases, in principle, have remarkable synthetic possibilities, but commerciai processes based on these enzymes have not yet been developed. The inactivation of heme- enzymes by peroxides through oxidation of the porphyrin ring is one of the prime reasoos. Attempts to improve the stability have not been very successful yet. The enzyme stability can be improved by stepwise or continuous addition of the oxidaot, maintaining a low peroxide concentration [<a href="#ref">2,3</a>]. Hiner et al. [<a href="#ref">4</a>] tried to increase the resistance of HRP to H <sub>2</sub> O <sub>2</sub> through genetic engineering without satisfying results. Other ways to deal with the problem of poor stability were chemical modification, screening and immobilisation [<a href="#ref">5</a>].</p><p>A second reason why commercial processes based on peroxidases have not yet been developed is that the oxidation of some substrates by peroxidases is in competition with their spontaneous chemical oxidation by peroxides. As a consequence, reduction of the purity of products occurs in case enantioselectivity is the target [<a href="#ref">6</a>]. Maintaming a low peroxide concentration throughout the reaction period, reducing background oxidation, has also been tried here as a solution.</p><p>Furthermore, in the process of one-electron oxidation by peroxidases free radicals are produced which are difficult to control, thereby reducing the purity of products as well.</p><p>A fourth reason for the limited scaiing up of peroxidase-catalysed reactions is the low water solubility of most of 8e substrates of synthetic interest. Designing strategies that enhance enzymatic activity in organic solvents [<a href="#ref">7,8</a>], or the use of hydrophobic matrices that act as a reservoir for both substrates and products are solutions to this general problem.</p><p>From the above it can be concluded that the problem of (operational) stability of peroxidases is a severe problem for industrial processes and that this problem is difficult to so)ve. On the other hand this thesis shows that a peroxidase exists that, in spite of its low operational stability, can be an efficient biocatalyst for the production of a industrially relevant compound with high yield and purity. Altogether, it can be concluded that peroxidases could represent an interesting tool for industrially relevant reactions, making future research on possibilities and limitations worthwhile.</p><h3><a name="ref">References</a></h3><ol><li>Hendriksen, A., Schuller, D.J., Meno, K., Welinder, K.G., Smith, A.T., Gaijhede, M. (1998) <em>Biochem.</em><strong>37</strong> : 8054-8060.</li><li>Colonna, S., Gaggero, N., Casella, L., Carrea, G., Pasta, P., (1992) <em>Tetrahedron asymmetry</em><strong>3</strong> : 95-106.</li><li>Van Deurzen, M.P.J., Van Rantwijk, F., Sheldon, R.A. (1996) <em>J. Mol. Catal. B. Enzym.</em><strong>2</strong> : 33-42</li><li>Hiner, A.N.P., Hernandez-Ruiz, J., Arnao, M.B., Garcia-Canovas, F., Acosta, M. (1996) <em>Biotechnol. Bioeng.</em><strong>50</strong> : 655-662</li><li>Bakker, M., Van de Velde, F., Van Rantwijk, F., Sheldon, R.A. (in preperation) <em>Biotechnol. Bioeng.</em></li><li>Colonna, S., Gaggero, N., Richelmi, C., Pasta, P. (1999) <em>Tibtech.</em><strong>17</strong> : 163-168</li><li>Klibanov, A.M., (1997) <em>Trends Biotechnol.</em><strong>15</strong> : 97-101</li><li>Osman, A.M., Boeren, S., Boersma, M.G., Veeger, C., Rietjens, I.M.C.M. (1997) <em>Proc. Natl. Acad. Sci. USA</em><strong>94</strong></li></ol>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Rietjens, Ivonne, Promotor
  • Laane, N.C.M., Promotor, External person
Award date21 Nov 2000
Place of PublicationS.l.
Publisher
Print ISBNs9789058082855
Publication statusPublished - 2000

Keywords

  • peroxidases
  • chemical structure
  • enzymes

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