Enzymes concentration

Michaelis constant An experimentally determined parameter inversely indicative of the affinity of an enzyme for its substrate. For a constant enzyme concentration, the Michaelis constant is that substrate concentration at which the rate of reaction is half its maximum rate. In general, the Michaelis constant is equivalent to the dissociation constant of the enzyme-substrate complex. 

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. 

The response of the immobilized enzyme electrode can be made independent of the enzyme concentration by using a large excess of enzyme at the electrode surface. The electrode response is limited by the mass transport of the substrate. Using an excess of enzyme often results in longer electrode lifetimes, increased linear range, reduced susceptibiUty to pH, temperature, and interfering species (58,59). At low enzyme concentrations the electrode response is governed by the kinetics of the enzyme reaction. 

The Michaelis-Menten scheme nicely explains why a maximum rate, V"max, is always observed when the substrate concentration is much higher than the enzyme concentration (Figure 11.1). Vmax is obtained when the enzyme is saturated with substrate. There are then no free enzyme molecules available to turn over additional substrate. Hence, the rate is constant, Vmax, and is independent of further increase in the substrate concentration. 

Equation 1-106 predicts that the initial rate will be proportional to the initial enzyme concentration, if the initial substrate concentration is held constant. If the initial enzyme concentration is held constant, then the initial rate will be proportional to the substrate concentration at low substrate concentrations and independent of the substrate concentration at high substrate levels. The maximum reaction rate for a given total enzyme concentration is... 

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. 

Equation (3-150) is the Michaelis-Menten equation, Vm is the maximum velocity (for the enzyme concentration ,), and is the Michaelis constant. 

Overall enzyme balance and equilibrium constants are defined for the intermediate substrate and enzyme complex. The total enzyme concentration is the sum of free and conjugated enzymes with the substrates. 

The initial and total enzyme concentrations are defined based on measurable components given below ... 

The total enzyme concentration is the sum of free and conjugated enzymes with substrates. 

From the equilibrium constant, the free enzyme concentration must be defined. We know the total enzyme concentration as the sum of the conjugated enzymes with substrates and the free enzymes. 

If [E]t is used to represent the total enzyme concentration, then [E]ss Thus Eq. (4-111) can be rearranged as... 

The rate is independent of the substrate concentration and first order with respect to enzyme concentration. In this case reaction (3), in which the complex decomposes to form the product, is the slowest step and is therefore rate limiting. Although this discussion has assumed that we have only an isolated enzyme reacting with a substrate, the same principles are applied to the more complex case when an entire organism, or a series of organisms consumes a substrate. 

Reaction 1 is the slowest step in this series of reactions leading to product formation. It is the rate-limiting step. Since this reaction involves bringing E and S together, it is a second-order reaction overall and first order with respect to the total enzyme concentration and the substrate concentration. 

Reactions proceed via transition states in which AGp is the activation energy. Temperature, hydrogen ion concentration, enzyme concentration, substrate concentration, and inhibitors all affect the rates of enzyme-catalyzed reactions. 

It is relevant also to compare the results in Fig.5 with previously published data for PAC production under similar environmental conditions, where with higher concentrations of initial benzaldehyde (600 mM), pyruvate (400 mM) and PDC activity (8.4 U ml ) a similar maximum concentration of PAC of 330 mM was produced [6]. PDC stability was similar in both processes with half life values of approximately 27h. However, PAC production was much faster in the benzaldehyde emulsion system, presumably due to higher initial enzyme concentration. 

It has been demonstrated that such controls are not valid and that there may be significant differences in enzyme concentrations between two cell types (56). For this reason, normal amniotic fluid cells themselves must be used as controls for amniotic fluid cell cultures being subjected to enzymologic inves t igat ion. 

Available methods provide measurements of enzyme activity rather than of enzyme concentration. In order that the measured activity be proportional to enzyme concentration, the reaction conditions which include pH, temperature, initial substrate concentration, sample and total volume and reaction time must be held constant and be carefully controlled. 

End-point methods are often not based on kinetic-ally optimum conditions. However, an end-point method is often the only convenient one available. In this case, the method should have been validated by showing that the catalysis of the substrate follows well defined kinetics, rate of reaction is proportional to enzyme concentration, blanks and interfering substances are corrected for, and that appropriate standards are available. 

The measured activity should be directly proportional to enzyme concentration over a practical working range. 

Amylase enters the blood largely via the lymphatics. An increase in hydrostatic pressure in the pancreatic ducts leads to a fairly prompt rise in the amylase concentration of the blood. Neither an increase in volume flow of pancreatic juice nor stimulation of pancreatic enzyme production will cause an increase in senm enzyme concentration. Elevation of intraductal pressure is the important determinant. Stimulation of flow in the face of obstruction can, however, augment the entry of amylase into the blood, as can disruption of acinar cells and ducts. A functional pancreas must be present for the serum amylase to rise. Serum amylase determination is indicated in acute pancreatitis in patients with acute abdominal pain where the clinical findings are not typical of other diseases such as appendicitis, cholecystitis, peptic ulcer, vascular disease or intestinal obstruction. In acute pancreatitis, the serum amylase starts to rise within a few hours simultaneously with the onset of symptoms and remains elevated for 2 to 3 days after which it returns to normal. The peak level is reached within 24 hours. Absence of increase in serum amylase in first 24 hours after the onset of symptoms is evidence against a diagnosis of acute pancreatitis (76). 

Several tubidimetric methods (92, 93) have been described specifically for the kinetic determination of lipase in serum but these methods suffer from lack of linearity with increasing enzyme concentration, and instability of emulsions. 

Because enzyme, substrate and product are all in the same medium we can conveniently work with concentrations. With the total enzyme concentration equal to [EJtot, the conservation of active species requires that... 

The turnover number was obtained as V/sq, where eg is the molar enzyme concentration. 

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