Unbound enzyme concentration

The total bound and unbound enzyme concentration, [E]o, is given by ... 

Wc still cannot use this rate law because we cannot measure the unbound enzyme concentration (E) however, we can measure the total enzyme concentration, Ef,... 

The total enzyme concentration is the sum of the bound and unbound enzyme concentrations... 

First of all, the behavior of the enzymes in the membrane differs markedly from the behavior of the unbound enzymes in solution. It is pertinent to note that the medium in which the enzyme bound to a membrane acts might be determined not only by the composition and structure of the membrane itself, but also by the local concentration distribution of substrate and products. The microenvironment in the membranes is the result of a balance between the flow of matter and enzyme reactions. The substrate and product concentrations in the membrane differ from point to point across the membrane and also from those at the outer solution. By electron microscopy this was experimentally demonstrated beyond doubt with the DAB-peroxidase system by Barbotin and Thomas.16 The effects of these profiles were studied with... 

Because [ES] in Equation 6-11 is not easily measured experimentally, we must begin by finding an alternative expression for this term. First, we introduce the term [Et], representing the total enzyme concentration (the sum of free and substrate-bound enzyme). Free or unbound enzyme can then be represented by [Et] — [ES]. Also, because [S] is ordinarily far greater than [Et], the amount of substrate bound by the enzyme at any given time is negligible compared with the total [S]. With these conditions in mind, the following steps lead us to an expression for V0 in terms of easily measurable parameters. 

The rate of reaction at low substrate concentration is proportional to the saturation rate, kB, and concentration of the array of enzyme-substrate complexes, which is proportional to l/Km thus, the rate is proportional to ks/Km. Since stabilization of the complex by changing pH, for example, increases the concentration of the complex at the same time as it decreases the probability of activation to a transition state, the net result is that k /Km is related to the free energy of the activated complex relative to the unbound enzyme, an unbound substrate. The pH profile of ke/Km thus potentially reveals the pK values of groups on the free enzyme and free substrate that are involved significantly in rate limiting processes. This well-known relationship has been used to establish the pIC values of 5.4 and 6.4 for groups on the enzyme in 0.2 M KC1 for both... 

Unfortunately, [ES] is difficult to measure during a reaction, so the rate equation needs to be written without this term. Michaelis and Menten recognized that [E], unbound enzyme, is equivalent to the total enzyme concentration, [Et], less [ES] (Equation 4.2). Therefore, the rate of [ES] formation is described in Equation 4.3. The rate of ES consumption can also be described in an equation (Equation 4.4). 

From the material balance, the total concentration of the enzyme in the system, CEX, is constant and equal to the sum of the concentrations of the free or unbounded enzyme, CE, and the enzyme-substrate complex, CSE, that is ... 

Another parameter often referred to when discussing Michaelis-Menten kinetics is kcaJ Ky. This is an apparent second-order rate constant that relates the reaction rate to the free (not total) enzyme concentration. As described above, at very low substrate concentrations when the enzyme is predominantly unbound, the velocity (f) is equal to [El Ky. The value of Is JKy sets a lower limit on the rate constant for the association of enzyme and substrate. It is sometimes referred to as the specificity constant because it determines the specificity of the enzyme for competing substrates. 

In two double-blind, randomized, placebo-controlled studies there was no inhibition of the metabolism of theophylline by azithromycin (53,54). However, there has been a report of reduced theophylline concentrations after withdrawal of azithromycin (55). The authors concluded that the mechanism of interaction was best explained by concomitant induction and inhibition of theophylline metabolism by azithromycin, followed by increased availability of unbound enzyme sites as azithromycin was cleared from the system. 

Homogeneous enzyme immunoassays have also been developed for serum T4 determination. These procedures are rapid and simple to use and have also been applied to several major automated instruments.For example, the enzyme-multiplied immunoassay technique (EMIT) for T4 measurement uses glucose-6-phosphate dehydrogenase covalently hnked to T4 as the enzyme label.Binding of T4 specific antibody to this label reduces enzyme activity, perhaps as a result of steric or allosteric inhibition As the concentration of unlabeled T4 increases, less enzyme-labeled hormone is bound by the antibody. As a result, the catalytic activity of the unbound enzyme conjugate increases in direct proportion to the amount of T4 in the specimen. The indicator reaction involves oxidation of glucose-6-phosphate with simultaneous reduction of nicotinamide-adenine dinu-... 

Using the PSSH for E S) and a balance on the total enzymt . E, w includes both the bound (E S) and unbound enzyme (E) concentrations... 

A typical photometric reaction is, for example, the interaction between hydrogen peroxide and 2-phe-nylenediamine, leading to the colored quinonedi-imine with an absorption maximum at 492 nm. This reaction is catalyzed by the above-mentioned peroxidase enzyme and, hence, the absorbance is proportional to the concentration of the unbound enzyme steroid complex in the equilibrium mixture. 

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