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Kinetics: Determination of an Enzymes Activity - Relevance

Proteins serve as biochemical catalysts in perhaps their greatest and most important role. Catalysts are substances that increase product formation by (1) lowering the energy barrier (activation energy) for the product to form

Enzyme = Catalyst

and (2) increases the favorable orientation of colliding reactant molecules for product formation to be successful.

A number of things can happen to throw a monkey wrench into this remarkably efficient process.

Temperature Effects on Enzyme Activity

Every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is rendered inactive and is said to be totally inhibited. This occurs because as the temperature changes this supplies enough energy to break some of the intramolecular attractions between polar groups (Hydrogen bonding, dipole-dipole attractions) as well as the Hydrophobic forces between non-polar groups within the protein structure. When these forces are disturbed and changed, this causes a change in the secondary and tertiary levels of protein structure, and the active site is altered in its conformation beyond its ability to accomodate the substrate molecules it was intended to catalyze. Most enzymes (and there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value which varies according to each individual. This can happen if body temperature gets too low (hypothermia) or too high (hyperthermia).

pH Effects on Enzyme Activity

Changes in the pH or acidity of the environment can take place that would alter or totally inhibit the enzyme from catalyzing a reaction. This change in the pH will affect the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule could no longer fit, and the chemical change would be inhibited from taking place as efficiently or not at all. In an acid solution any basic groups such as the Nitrogen groups in the protein would be protonated. If the environment was too basic the acid groups would be deprotonated. This would alter the electrical attractions between polar groups. Every enzyme has an optimum pH range outside of which the enzyme is inhibited. Some enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. Other enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near neutrality. Still other enzymes like the lipases will function most effectively at basic pH values. If the pH drops in the blood called acidosis then enzymes in the blood will be inhibited outside their optimal pH range. If the pH climbs to an unacceptably high value called alkalosis then enzymes cease to function effectively. Normally, these conditions do not take place because of the highly efficient buffers found in the blood that restrict the pH of the blood to a very narrow range. Buffers are a substance or mixtures of substances that resist any change in the pH. There are many buffer systems found in the body to adjust the pH so that enzymes might continue to catalyze their reactions.

Correcting pH or temperature imbalances will usually allow the enzyme to resume its original shape or conformation. Some substances when added to the system will irreversably break bonds disrupting the primary structure so that the enzyme is inhibited permanently. The enzyme is said to be irreversably denatured. Many toxic substances will break co-valent bonds and cause the unraveling of the protein enzyme. Other toxic substances will precipitate enzymes effectively removing them from the solution thus preventing them from catalyzing the reaction. This is also called denaturation.

Substrate Concentration Effects on Enzyme Activity

It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). This is represented graphically in Figure 8.

ie13

It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this maximum velocity and equation (1), Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data.

ie08 (1)

The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is shown in Figure 8. Using this constant and the fact that Km can also be defined as:

ie14

K+1, K-1 and K+2 being the rate constants from equation (1). Michaelis developed the following:


Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme.

  • A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.
  • A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.
  • The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes.