Kinetic high enzyme concentrations

One of the earliest demonstrations by ESR of the peroxidase oxidation of drugs involved the tranquilizer, chlorpromazine [171]. The stoichiometry of the reaction forming the chlorpromazine radical cation (13) was established, and kinetics of the transient radical were shown to be identical to those of an optically detected intermediate absorbing at 530 nm. Measurements were made of the pH-dependent radical dismutation rate and the rate of reaction of compound II with chlorpromazine. At high enzyme concentrations and low dismutation rates, the radical cation undergoes further oxidation by the enzyme, to form the thionium ion. 

From the kinetics of the enzymatic and the non-enzymatic reactions (Fig. 7-13) it is concluded that the side-reaction is suppressed very effectively by working with high enzyme concentrations and at a low benzaldehyde concentration. Benzalde-hyde may react with amino functions of the enzyme to form Schiffbases resulting in deactivation of oxynitrilase, so low stationary benzaldehyde concentrations are also necessary with respect to enzyme stability. 

Ultrafiltration of an enzyme solution through a UF membrane does not always result in gel layer formation. Unless a gel layer is formed, this immobilization technique cannot be used for flow systems lacking effective enzyme immobilization. In any event, soluble enzyme membrane reactors can be useful in order to perform kinetic analysis at high enzyme concentrations. Once steady state is attained, the theoretical model permits calculation of reaction rates in both regions. Once the layer is formed it behaves like a secondary membrane,34 capable of separating compounds of different molecular weight in the mixture as well as catalyzing a chemical reaction. 

The stopped-flow rapid-mixing and temperature-jump kinetic studies from the author s laboratory (72,89) which describe the reaction of the enzyme-NADH complex with the intense chromophore, direct evidence for the involvement of zinc ion as a Lewis acid in the activation of the aldehyde carbonyl for reduction. These studies show that reduction of DACA involves the formation of a transient chemical intermediate (Amax 464 nm, emax 6.2 X 10 M-icm i) in the neutral pH range. At pH values above 9 and in the presence of high enzyme concentrations, the transient species observed... 

Eor measurement of a substrate by a kinetic method, the substrate concentration should be rate-limiting and should not be much higher than the enzyme s K. On the other hand, when measuring enzyme activity, the enzyme concentration should be rate-limiting, and consequentiy high substrate concentrations are used (see Catalysis). 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

Kinetic studies involving enzymes can principally be classified into steady and transient state kinetics. In tlie former, tlie enzyme concentration is much lower tlian that of tlie substrate in tlie latter much higher enzyme concentration is used to allow detection of reaction intennediates. In steady state kinetics, the high efficiency of enzymes as a catalyst implies that very low concentrations are adequate to enable reactions to proceed at measurable rates (i.e., reaction times of a few seconds or more). Typical enzyme concentrations are in the range of 10 M to 10 ], while substrate concentrations usually exceed lO M. Consequently, tlie concentrations of enzyme-substrate intermediates are low witli respect to tlie total substrate (reactant) concentrations, even when tlie enzyme is fully saturated. The reaction is considered to be in a steady state after a very short induction period, which greatly simplifies the rate laws. 

The kinetics of enzyme reactions were first studied by the German chemists Leonor Michaelis and Maud Menten in the early part of the twentieth century. They found that, when the concentration of substrate is low, the rate of an enzyme-catalyzed reaction increases with the concentration of the substrate, as shown in the plot in Fig. 13.41. However, when the concentration of substrate is high, the reaction rate depends only on the concentration of the enzyme. In the Michaelis-Menten mechanism of enzyme reaction, the enzyme, E, and substrate, S, reach a rapid preequilibrium with the bound enzyme-substrate complex, ES ... 

This equation is fundamental to all aspects of the kinetics of enzyme action. The Michaelis-Menten constant, KM, is defined as the concentration of the substrate at which a given enzyme yields one-half of its maximum velocity. is the maximum velocity, which is the rate approached at infinitely high substrate concentration. The Michaelis-Menten equation is the rate equation for a one-substrate enzyme-catalyzed reaction. It provides the quantitative calculation of enzyme characteristics and the analysis for a specific substrate under defined conditions of pH and temperature. KM is a direct measure of the strength of the binding between the enzyme and the substrate. For example, chymotrypsin has a Ku value of 108 mM when glycyltyrosinylglycine is used as its substrate, while the Km value is 2.5 mM when N-20 benzoyltyrosineamide is used as a substrate... 

The expression for the effectiveness factor q in the case of zero-order kinetics, described by the Michaelis-Menten equation (Eq. 8) at high substrate concentration, can also be analytically solved. Two solutions were combined by Kobayashi et al. to give an approximate empirical expression for the effectiveness factor q [9]. A more detailed discussion on the effects of internal and external mass transfer resistance on the enzyme kinetics of a Michaelis-Menten type can be found elsewhere [10,11]. 

Figure 11.1 illustrates the behavior of Equation 11.6. By the assumption of rapid equilibrium the rate determining step is the unimolecular decomposition. At high substrate composition [S] KM and the rate becomes zero-order in substrate, v = Vmax = k3 [E0], the rate depends only on the initial enzyme concentration, and is at its maximum. We are dealing with saturation kinetics. The most convenient way to test mechanism is to invert Equation 11.6... 

In cases where the depuration of HOCs from BMOs involves enzyme-mediated biotransformations (Eq. 7.4) or active transport mechanisms, and environmental concentrations are high (e.g. near a point source), depuration rates have been shown to follow Michaelis-Menten kinetics (Spade and Hamelink, 1985). Michaelis-Menten kinetics is elicited when an enzyme or active transport system is saturated with a chemical. This type of kinetics is characterized by lower values of keS at sites with high HOC concentrations. If k s are unchanged at high concentration sites, Michaelis-Menten kinetics will result in elevated BAFs. However, if chemical concentrations become toxic, finfish likely avoid the area and sessile organisms such as mussels may close their valves for extended periods (Huckins et al., 2004). 

The first two reported studies concern the epoxide hydrolase from Aspergillus niger (ANEH) 95,96). The enzyme had previously been purified to homogeneity, the gene cloned and expressed in E. coli, and the catalytic hydrolysis of epoxides optimized to high substrate concentrations. Initial attempts were made to enhance the enantioselectivity of the ANEH-catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-33). The WT leads to an E value of only 4.6 in favor of (5)-34 96). 

There are quite a few situations in which rates of transformation reactions of organic compounds are accelerated by reactive species that do not appear in the overall reaction equation. Such species, generally referred to as catalysts, are continuously regenerated that is, they are not consumed during the reaction. Examples of catalysts that we will discuss in the following chapters include reactive surface sites (Chapter 13), electron transfer mediators (Chapter 14), and, particularly enzymes, in the case of microbial transformations (Chapter 17). Consequently, in these cases the reaction cannot be characterized by a simple reaction order, that is, by a simple power law as used for the reactions discussed so far. Often in such situations, reaction kinetics are found to exhibit a gradual transition from first-order behavior at low compound concentration (the compound sees a constant steady-state concentration of the catalyst) to zero-order (i.e., constant term) behavior at high compound concentration (all reactive species are saturated ) ... 

---