Enzymes turnover frequency

The shape of the hydrogenase catalytic voltammograms shown in Fig. 17.14 also changes as the temperamre is raised. At 10 °C, the current tends towards a plateau at high overpotential as catalysis becomes limited by the inherent turnover frequency of the enzyme, but at higher temperamres, the current continues to increase linearly with electrochemical driving force. This has been attributed to a range of different... 

In addition to their proven capacity to catalyze a highly efficient and rapid reduction of O2 under ambient conditions (e.g., cytochrome c oxidase, the enzyme that catalyzes the reduction of >90% of O2 consumed by a mammal, captures >80% of the free energy of ORR at a turnover frequency of >50 O2 molecules per second per site), metalloporphyrins are attractive candidates for Pt-free cathodes. Probably the major impetus for a search for Pt-free cathodic catalysts for low temperature fuel cells is... 

The turnover frequency, N, (commonly called the turnover number) defined, as in enzyme catalysis, as molecules reacting per active site in unit time, can be a useful concept if employed with care. In view of the problems in measuring the number of active sites discussed in 1.2.4, it is important to specify exactly the means used to express Q in terms of active sites. A realistic measure of such sites may be the number of surface metal atoms on a supported catalyst but in other cases estimation on the basis of a BET surface area may be the only readily available method. Of course, turnover numbers (like rates) must be reported at specified conditions of temperature, initial concentration or initial partial pressures, and extent of reaction. 

The turnover frequency allows performance comparison between different catalyst systems, biological and/or non-biological. Its threshold is at 1 event per second per active site. According to the definition, a turnover frequency can be determined only if the number of active sites is known (Chapter 9, Section 9.2.3). For an enzyme reaction obeying Michaelis-Menten kinetics, Eq. (2.15) holds. 

V max has limited utility because it is related to [Et], an experimentally controlled variable. In contrast, k2 is a property of the enzyme. For the reaction model in Scheme 4.7, k2 is the rate-determining step in the catalytic process and is frequently labeled kcaV Like k2, kcat is a first-order rate constant with units of inverse time. kcat is often called the turnover frequency (TOF), the maximum number of substrate molecules an enzyme can convert to product per unit time. For an enzyme to achieve a maximum kcat, reaction conditions, namely temperature and pH, must be optimal. 

Finally, but certainly not least, we note that the enzyme chloroperoxidase (CPO), catalyzes the highly enantioselective (>98% ee) sulfoxidation of a range of substituted thioanisoles [308]. In contrast to the epoxidation of alkenes, where turnover frequencies were low (see above), in the case of sulfoxidation of thioa-nisole a turnover frequency of around 16 s-1 and a total turnover number of 125000 could be observed. A selection of data is represented in Fig. 4.112. Besides aryl alkyl sulfides, also dialkylsulfides could be oxidized with reasonable enantioselectivities [27]. 

Another class of peroxidases which can perform asymmetric sulfoxidations, and which have the advantage of inherently higher stabilities because of their non-heme nature, are the vanadium peroxidases. It was shown that vanadium bromoperoxidase from Ascophyllum nodosum mediates the production of (R)-methyl phenyl sulfoxide with a high 91% enantiomeric excess from the corresponding sulfide with H202 [38]. The turnover frequency of the reaction was found to be around 1 min-1. In addition this enzyme was found to catalyse the sulfoxidation of racemic, non-aromatic cyclic thioethers with high kinetic resolution [309]. 

Similarly, when vanadate is introduced in the active site of the acid phosphatases, these artificial enzymes are able to oxidize bromide in the presence of H2O2 and to sulfoxidize sulfides in an enantioselective manner. The turnover frequency... 

Hassler et al. [40, 41] observed that the stretched exponential behavior was related to the active phase of the enzyme (busy phase) while the less active phase (lazy phase) exhibited a single exponential phase (Fig. 4.20). The behavior which was originally observed by Edman et al. 1999 [39] at a much lower sensitivity had been observed again, however, with a much better intensity and time resolution. As can be seen from the trace, the turnover frequency is more than 4-fold higher in the busy periods. It is tempting to relate this to the memory effect decribed in the next chapter (Fig. 4.21). 

In another approach, Yoneyama used electrogenerated and regenerated 1-phenyl-ethanol as reducing equivalent for the alcohol dehydrogenase-catalyzed regeneration of NADH. In this system, the production enzyme and the cofactor regenerating enzyme are identical. A production of 2 pmol was reached at turnover frequency of 1.7 TN/h or less (Fig. 9) [46]. 

The value for V, ax 27" (mole site) , is the maximum rate for this reaction when it is run under saturation conditions for both substrates. In contrast to the single type of active site found in most enzymes, there are a number of different types of sites present on the surface of the platinum catalyst used for these oxidations. It was shown in a parallel study that 2-propanol oxidation takes place over the coordinately unsaturated corner atoms, that is, the single turnover (STO) characterized M, and MH sites (see Chapter 2). It was also shown that the specific site turnover frequencies (TOF) for these sites are 5.5,7.9 and 5.0 moles O2 uptake/mole site/minute respectively. 

Fig. 2.11 Enzyme-catalyzed desymmetrization of meso-epoxides using epoxide hydrolases of microbial origin that were screened for maximum activity. Note that the relative rate (measured as the turnover frequency, TOE) offormingthe (R,R)-stereoisomer is about 250 times higher than for the (S,S) one.

It follows that if an enzyme has a very high turnover frequency, it may be difficult to determine its inherent catalytic properties. The [NiFe]-hydrogenase from Allochromatium vinosum is a good example. A film of this enzyme formed on a PGE electrode displays very high activity for Hz oxidation and, for an atmosphere of 10% Hz, the catalytic current varies with... 

Several co-oxo fatty acids are transformed to the corresponding a,co -dicarboxylic acids, whereas -formylesters of fatty acids are decarboxylated to the co-hydroxy fatty acids and carbon dioxide1111. For several co-oxo fatty acids turnover frequencies (measured as O2 consumption) between 1.8 to 25 s 1 were found. Many P450 systems are multi-component enzymes with small protein cofactors such as putidaredoxin performing the electron mediation between NAD(P)H and the active site of the enzyme. Vilker and coworkers recently were able to show that NADPH can be omitted from the catalytic cycle by direct electrochemical reduction of putidar-... 

PrCL) and 6-n-butyl-e-caprolactone (6-BuCL) were synthesised and subjected to a Novozym 435 catalysed ring-opening. The turnover frequencies (TOP) and the enantiomeric ratio E, which is a measure of the enantioselectivity of the enzyme for the lactone, were derived from the kinetic plots. 

The turnover frequency (TOP) is defined as the number of turnovers per enzyme molecule per second at the start of reaction. In order to calculate this number, an active protein content of 10 wt.% of the immobilized preparation is assumed. The TOP is calculated using the formule TOP = ki (initial substrate concentration) / (total enzyme concentration). The initial rate constant (ki) is the slope of the ln(l-conversion) versus time plot. 

The turnover frequency TOF (the term was borrowed from enzyme catalysis) quantifies the specific activity of a catalytic center for a special reaction under defined reaction conditions by the munber of molecular reactions or catalytic cycles occuring at the center per unit time. For heterogeneous catalysts the number of active centers is derived usually from sorption methods (Eq. 1-7). 

Kinetics of enzyme-catalyzed reactions may often be reported as a turnover frequency. Explain this term. 

The interpretation follows from the limiting case of Equation 4-8. Consider the limiting case of a high reactant concentration which is so high that the catalytic sites are saturated and [5 ] K - Then, the rate equation reduces to = cat[ ]tot and kcat is recognized as a first-order rate constant. If the rate were written per enzyme molecule rather than per unit volume, then the reaction would be of zero order, and kcat would be the rate at saturation (the maximum number of reactant molecules converted per catalytic site per unit time) this is the definition of the turnover frequency. 

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