Enzyme activity substrate concentration affecting

FACTORS AFFECTING ENZYME ACTIVITY Substrate concentration... 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

Factors that affect the rate of enzyme-catalyzed reactions include enzyme and substrate concentration, pH, temperature, and the presence of inhibitors, activators, co-enzymes, and prosthetic groups. 

No enzymatic side effects are observed and substrate concentrations up to 20% by weight can be used without affecting the enzyme activity. The biocatalyst is used in soluble form in a batch wise process, thus poorly soluble amino adds can be resolved without technical difficulties. Re-use of the biocatalyst is in prindple possible. 

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. 

At very low substrate concentration ([S] approaches zero), the enzyme is mostly present as E. Since an uncompetitive inhibitor does not combine with E, the inhibitor has no effect on the velocity and no effect on Vmsa/Km (the slope of the double-reciprocal plot). In this case, termed uncompetitive, the slopes of the double-reciprocal plots are independent of inhibitor concentration and only the intercepts are affected. A series of parallel lines results when different inhibitor concentrations are used. This type of inhibition is often observed for enzymes that catalyze the reaction between two substrates. Often an inhibitor that is competitive against one of the substrates is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor does combine at the active site but does not prevent the binding of one of the substrates (and vice versa). 

As discussed earlier, the tacit assumption of in vitro studies is that they are faithful reporters of how the enzymes and substrates will behave in vivo. At least qualitatively, the assumption seems largely to be true but quantitatively the assumption is less reliable. It assumes that the different microenvironments surrounding an enzyme in vivo and in an in vitro preparation do not differentially affect kinetic properties. It also assumes that, given equal concentrations of drug, the concentration that actually reaches the active site of the enzyme in the two different microenvironments will be equal (5). Clearly this does not need to be the case. As a consequence, a more reliable reporter of the in vivo kinetic properties of a drug would be highly desirable. 

A very useful complement to enzyme assays as described above is histochemical study, which can provide additional information [76]. In particular, because it is possible to measure the activity cell per cell, histochemistry permits, in the case of a heteroplasmic population of mitochondria, the detection of even a small number of affected cells, which may have remained undetected by biochemical assays. Spectacular images showing, side-by-side, cells endowed with either high or absent enzyme activity can be obtained. The limitation of the method is in part due to the few activities possibly measured (essentially complex IV, succinic dehydrogenase, and less specifically, ATPase and NADH reductase) and to the fact that it is poorly quantitative. Histochemical investigations are performed under selected conditions (e.g., substrate concentrations, pH), which often differ from those used for enzyme assays, thus possibly introducing discrepancy between the two approaches. 

Most, if not all, milks contain sufficient amounts of lipase to cause rancidity. However, in practice, lipolysis does not occur in milk because the substrate (triglycerides) and enzymes are well partitioned and a multiplicity of factors affect enzyme activity. Unlike most enzymatic reactions, lipolysis takes place at an oil-water interface. This rather unique situation gives rise to variables not ordinarily encountered in enzyme reactions. Factors such as the amount of surface area available, the permeability of the emulsion, the type of glyceride employed, the physical state of the substrate (complete solid, complete liquid, or liquid-solid), and the degree of agitation of the reaction medium must be taken into account for the results to be meaningful. Other variables common to all enzymatic reactions—such as pH, temperature, the presence of inhibitors and activators, the concentration of the enzyme and substrate, light, and the duration of the incubation period—will affect the activity and the subsequent interpretation of the results. 

Equations (5) and (10) imply that the velocity of an uncatalyzed reaction increases indefinitely with an increase in the concentration of the reactants. With enzyme-catalyzed reactions, something very different is observed. The rate usually increases linearly with substrate concentration at low concentrations, but then levels off and becomes independent of the concentration at high concentrations (fig. 7.6). The explanation for this hyperbolic dependence on substrate concentration is straightforward. For an enzyme to affect AG, the substrate must bind to a special site on the protein, the active site (fig. 7.7). At very low concentrations of substrate, the active sites of most of the enzyme molecules in the solution are unoccupied. Increasing the substrate concentration brings more enzyme molecules into play, and the reaction speeds up. At high concentrations, on the other hand, most of the enzyme molecules have their active sites occupied, and the observed rate depends only on the rate at which the bound reactants are converted into products. Further increases in the substrate concentration then have little effect. 

The effect of temperature satisfies the Arrhenius relationship where the applicable range is relatively small because of low and high temperature effects. The effect of extreme pH values is related to the nature of enzymatic proteins as polyvalent acids and bases, with acid and basic groups (hydrophilic) concentrated on the outside of the protein. Finally, mechanical forces such as surface tension and shear can affect enzyme activity by disturbing the shape of the enzyme molecules. Since the shape of the active site of the enzyme is constructed to correspond to the shape of the substrate, small alteration in the structure can severely affect enzyme activity. Reactor s stirrer speed, flowrate, and foaming must be controlled to maintain the productivity of the enzyme. Consequently, during experimental investigations of the kinetics enzyme catalyzed reactions, temperature, shear, and pH are carefully controlled the last by use of buffered solutions. 

---