Enzymatic reactions rates

The availability of substrates and cofactors will determine the enzymatic reaction rate. In general, enzymes have evolved such that their values approximate the prevailing in vivo concentration of their substrates. (It is also true that the concentration of some enzymes in cells is within an order of magnitude or so of the concentrations of their substrates.)... 

Compare the two cases in Eigure 16.3. Because the enzymatic reaction rate is determined by the difference in energies between ES and EX, the smaller... 

Today a good understanding of transition state structure can be obtained through a combination of experimental measurements of kinetic isotope effects (KIE) and computational chemistry methods (Schramm, 1998). The basis for the KIE approach is that incorporation of a heavy isotope, at a specific atom in a substrate molecule, will affect the enzymatic reaction rate to an extent that is correlated with the change in bond vibrational environment for that atom, in going from the ground state to the... 

In kinetic studies of enzymatic reactions, rate data are usually tested to determine if the reaction follows the Michaelis-Menten model of enzyme-substrate interaction. Weetall and Havewala [Biotechnol. and Bioeng. Symposium 3 (241), 1972] have studied the production of dextrose from cornstarch using conventional... 

The evaluation of the enzymatic reaction rates and how they are affected by the variation of experimental parameters can be of great help in understanding the mechanism of enzyme-catalyzed reactions. 

Although RMs are thermodynamically stable, they are highly dynamic. The RMs constantly colhde with each other and occasionally a colhsion results in the fusion of two RMs temporarily. During this fusion surfactant molecules and the contents residing inside RMs may be exchanged. In AOT reverse micellar system, this dynamic behavior exhibits second-order kinetics with rate constants in the order of 10 to 10 M s [37]. This dynamic nature not only influences the properties of the bulk system but also affects the enzymatic reaction rates [38]. 

Figure 2.3 Comparison of the relationship of KTS with the enzymatic reaction rate constant, kcat, and with the uncatalyzed solution or reference reaction rate constant kmn (Bruice, 2000).

In addition to exerting an influence on enzymatic reaction rates and the character of metabolism, temperature can also be a determining factor in the composition and conformation of biomolecules. 

Contrary to simple homogeneous catalytic processes, the temperature dependence of the enzymatic reaction rate shows growth in a narrow temperature range (temperature optimum) [86],... 

In biphasic reactors or two-phase partitioning bioreactors (TPPB), the substrate is located mostly in the immiscible phase and diffuses to the aqueous phase. The enzyme catalyzes conversion of the substrate at the interface and/or in the aqueous phase. The product/s of the reaction then may partition to the organic phase. The system is self-regulated, as the substrate delivery to the aqueous phase is only directed by the partitioning ratio between the two phases and the enzymatic reaction rate [53]. The use of ionic liquid/supercritical carbon dioxide for enzyme-catalyzed transformation is gaining attention [69]. 

Calorimetry. Isothermal titration calorimetry (ITC) has recently been proposed as a general method for the determination of enzymatic reaction rates.20 This method is based on the relationship between the power needed to maintain constant temperature and the number of moles of substrate converted. Power, the measured quantity, is related to heat (Q) by differentiation ... 

Thus, the measured thermal power for an enzyme reaction may be directly related to the enzymatic reaction rate ... 

The effects of this concentration gradient are most significant at low bulk concentrations of the substrate, since substrate is converted to product as soon as it reaches the surface of the particle, so that the surface concentration of substrate is zero. At very high bulk substrate concentrations, the enzymatic reaction rate is limited by enzyme kinetics rather than mass transport, so that surface concentrations do not differ significantly from those in the bulk. Because of the concentration gradient, however, enzyme saturation with substrate occurs at much higher bulk substrate concentrations than required to saturate the soluble enzyme. Apparent Km values (K m) for immobilized enzymes are larger than Km values obtained for the native soluble enzymes. 

In the enzymatic reaction rate diglyceride assay for LPS, the following sequence of indicator and auxiliary enzymes is used ... 

Waste oils, from restaurants and household disposals and being creating serious problems of environmental control and food safety, have been considered as good raw material for biodiesel production. Immobilized Candida antarctica lipase was found to be effective for the methanolysis of waste oil. A three-step methanolysis protocol could be used to protect lipase from inactivation by methanol. Compared with one-step reaction, it needs a longer time to reach the reaction equilibrium. So, efforts should be made to increase enzymatic reaction rate. Reports on the enhancement of the activity of certain enzymes by applying ultrasonic irradiation on the enzymes led us to investigate its effects on the enzymatic transesterification of waste oil to biodiesel in a solvent free system. 

This is a technological constraint, where the summation covers all enzyme fluxes (z in number, 30 in our case), nref is the i reference enzymatic reaction rate given in Table 13.1. Total enzymatic activity is constrained not to exceed 1.0 to avoid diffusion problem (due to increased cytoplasm viscosity), protein precipitation, secondary kinetic effects (due to steric hindrance) and excessive intracellular stress leading to unpredictable regulatory effects. When either one of these two constraints is breached, the objective function value is penalized by setting it to an arbitrarily low level under such conditions, the DAHPS, PEPCxylase and SerSynth fluxes are set to 10 °. 

Chi the basis of E. values we can make a conclusion about the difhision factors which are some of the most conqilicated points concerning catalysis with immobilized enzymes. The value for the activation energy on peroxidase oxidation of phenol with catalase immobilized on "NORIT" soot is E, =10.95 kJ.mof which is an indication that the process takes place under diSusion regime. The latter means that the enzymatic reaction rate is determined by the mass tranfer of substrate to the surfoce of the carrier particles and its diffiision into the carrier. 

The nature of the aqueous buffer (0.1 M NaCl, 4 mM sodium phosphate buffer pH 8.5 versus 0.1 M NaCl, 4 mM sodium borate buffer pH 8.5) had no appreciable effect on the enzyme selectivity although the hydrolysis was slightly faster with the phosphate. The buffer composition was simplified by omitting sodium chloride, while at the same time the phosphate concentration was increased to 10 mM in order to compensate for the ionic strength and increase the buffering capacity of the system. The pH value was kept at 8.5 as the optimal compromise between highest enzymatic reaction rate and lowest reaction rate of the unspecific alkaline ester hydrolysis. 

Richard (1984) has determined that TIM accelerates the rate of GAP to DHAP isomerization by almost 10 orders of magnitude over the rate enhancement provided by a simple base catalyst such as the acetate ion. Moreover, as indicated by the free energy profile, the enzymatic reaction rate is very fast in physical-chemical terms. kcat/Km (the pseudo first-order rate constant for the reaction of enzyme with substrate) in the thermodynamically favourable direction GAP to DHAP is 400,000,000 M Is 1, which is close to the expected diffusion-controlled limit. A Ferrari indeed ... 

More recently, confocal fluorimetry itself has been impressively extended. In particular, the implementation of multi-photon excitation opened the potential to excite different fluorescent labels by a single laser line [47]. This considerably simplified the optical setup of confocal instruments. For example, Heinze et al. [48] described a setup for two-photon excitation confocal fluorimetry where three molecular species were quantified simultaneously using a single laser. When included in screening systems, these spectroscopic advancements enable the quantification of enzymatic reaction rates on several substrates in parallel or, when applied for peptide or protein ligands, the simultaneous measurement of binding affinities on different target receptors. In this way, biopharmaceuticals can be selected on the basis of their specificity and selectivity. As a consequence, undesired side activities can be controlled very early in the hit identification process. 

A compilation of reported optimum water concentrations for maximum enzymatic reaction rates in SCCO2 is given in Table 4.9-4. 

Apart from the direct conformational changes in enzymes, which may occur at very high pressures, pressure affects enzymatic reaction rates in SCFs in two ways. First, the reaction rate constant changes with pressure according to transition stage theory and standard thermodynamics. Theoretically, one can predict the effect of pressure on reaction rate if the reaction mechanism, the activation volumes and the compressibility factors are known. Second, the reaction rates may change with the density of SCFs because physical parameters, such... 

Thus, there is experimental evidence that changing pressure actually changes enzymatic reaction rates at constant substrate concentrations (mol/volume). If, in a constant volume reactor, the substrate concentration (mol/volume) is kept constant and pressure rises, the mole fraction of substrates decreases. In almost all reported cases the pressure increase parallels the reaction rate decrease and the mol/mol concentration decrease. 

The experimental data that have accumulated since ca. 1992 have greatly increased the understanding of the interactions between enzymes and SCFs. It is now known that enzymatic reaction rates, substrate selectivity and enantiomeric selectivity can be tailored by altering the type and the density of the SCF. Although CO2 would be the most desirable industrial SCF as reaction medium, it is not suitable for all enzymes. It is an inhibitor for some proteases and lipases [20] but there are other common enzymes, including lipases, which are stable and active in SCCO2. Supercritical hydrocarbons and fluoroform are good and tunable solvents for enzymatic reactions. 

Even in SCFs, the enzymatic reaction rates are low compared with inorganic catalysis. Solute concentrations in SCFs also remain lower than in organic solvents. It is thought that enzymatic reactions in SCFs might be commercially utilized in fine chemical synthesis where the high specificity of enzymatic catalysis is required and where the production is limited to a few tons per year. Pharmaceutical producers are the most probable users of the technique. 

Eq. 3.6 represents the parametric expression for the enzymatic reaction rate as a function of substrate concentration. 

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