The Effect of Temperature on Protease.

Elmaz Korimbocus

The Effect of Temperature on Protease

Properties of Enzymes
As the Swedish chemist Jöns Jakob Berzelius suggested in 1823, enzymes are typical catalysts: they are capable of increasing the rate of reaction without being consumed in the process.

Some enzymes, such as pepsin and trypsin, which bring about the digestion of meat, control many different reactions, whereas others, such as urease, are extremely specific and may accelerate only one reaction. Still others release energy to make the heart beat and the lungs expand and contract. Many facilitate the conversion of sugar and foods into the various substances the body requires for tissue-building, the replacement of blood cells, and the release of chemical energy to move muscles.
Pepsin, trypsin, and some other enzymes possess, in addition, the peculiar property known as autocatalysis, which permits them to cause their own formation from an inert precursor called zymogens. As a consequence, these enzymes may be reproduced in a test tube.

As a class, enzymes are extraordinarily efficient. Minute quantities of an enzyme can accomplish at low temperatures what would require violent reagents and high temperatures by ordinary chemical means. About 30g of pure crystalline pepsin, for example, would be capable of digesting nearly 2 metric tons of egg white in a few hours.

The kinetics of enzyme reactions differs somewhat from those of simple inorganic reactions. Each enzyme is selectively specific for the substance in which it causes a reaction and is most effective at a temperature peculiar to it. Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated. The catalytic activity of an enzyme is determined primarily by the enzyme's amino-acid sequence and by the tertiary structure-that is, the three-dimensional folded structure of the macromolecule. Many enzymes require the presence of another ion or a molecule called a cofactor, in order to function.

As a rule, enzymes do not attack living cells. As soon as a cell dies, however, enzymes that break down protein rapidly digest it. The resistance of the living cell is due to the enzyme's inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, known as anti-enzymes, which prevent the action of an enzyme upon a substrate.

What holds enzymes together in the first place: Enzymes are proteins and proteins are constructed from chains of amino acids (20 possible different ones). The analogy of the amino acids being like links in a chain is a good one, although it should be visualised as having 20 different types of links, all that have different shapes and properties. As an unfolded chain, the enzyme has no catalytic activity. Only the folded structure forms the catalytic or active site. However this folded structure will generally be held together by non-covalent interactions unlike the covalent bonds that hold the amino acid links together. These consist of interactions such as ionic (salt) bridges, hydrogen bonds, hydrophopic and hydrophilic interactions and so on, which you will have come across in basic chemistry.

The Role of Ezymes in Biological Reactions

The laws of thermodymatics apply to chemical reactions anywhere in the universe. No reactions can violate the rules of thermodymatics. All reaction must proceed to a level of minimum energy and maximum entropy or have a favorable balance between the two. Enzyme simply increase the rate (rate is equal to the number of reactant molecules converted to product per unit of time) at which spontaneous reactions take place. Enzymes cannot make a reaction occur that would not proceed spontaneously without the enzyme. The same principle apply to reversible reaction. Enzymes do not alter the equilibrium point of reversible reaction.

Most biological reactions would take place to slow without enzyme for life to exist. The rates would be essentially zero at biological temperatures. For example the oxidation of glucose to CO2 and H2O is spontaneous and proceeds almost completely in the direction stated. However, without enzymes, glucose oxidation occurs do slowly at physiological temperatures that the rate is essentially unmeasureable.

The increases in rate achieve by enzymes, depending on the enzyme and reaction, range from a minimum of about a million to as much as a trillion times faster than the uncatalyzed reactions at equivalent concentrations and temperatures.

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Enzyme and Activation Energy

Activation energy is the energy barrier over which the molecules in a system must be raised for a reaction to take place (Figure 1).

This condition is analogous to a rock resting in a depression at the top of a hill. As long as the rock remains undisturbed, it will not spontaneously roll downhill unless activation energy is applied to the rock. Spontaneous movement over the barrier occurs because molecules, unlike the rock are in constant motion at temperatures above absolute zero. Although the average amount of movement, or kinetic energy, is below the amount required for activation, some molecular collision may raise a number of molecules to the energy level required for the reaction to proceed. The higher the activating barrier, the fewer the molecules that will proceed over the energy barrier per unit time.

Characteristics of Enzymes

Enzymes are protein molecules that are tailored to recognize and bind specific reactants and speed their conversion into products. These proteins are responsible for increasing the rates of all of the many thousand of reaction taking place inside cells.

All enzymatic proteins have several characteristics in common table 1.

Table 1: Characteristics of enzymes proteins

The rate of combination and release, known as the turnover number, lies near 1000 per second for most enzymes. Some enzymes have turnover numbers as small as 100 per second or as large as 10 million per second. As a result of enzyme turnover, a relatively small number of enzyme molecules can catalyze a large number of reactant molecules.

The part of an enzyme that combines with substrate molecule is the active site. In most enzymes the active site is located in a cavity or pocket on the enzyme surface, frequently within a cleft marking the boundary between two or more major domains. Within the cleft or pocket, amino acid side groups are situated to fit and bind parts of substrate molecules that are critical to the reaction catalyzed by the enzyme. The active site also separates substrate molecules from the surrounding solutions and place them in environments with unique characteristics, including partial or complete exclusion of water.

How Enzymes Lower the Energy of Activation

The mechanisms by which enzymes lower the energy of activation are still not totally understood. However, the mechanisms are believed to be directly or indirectly related to achievement of what is known as the transition state for a reaction. During any chemical interaction the reactants briefly enter a state in which old chemical bonds are incompletely broken and new ones are incompletely formed. In this transition state electron orbital assume intermediate positions between their locations in the reactants and their positions in the products. The transition state is highly unstable and can easily move in either direction with little change in energy - forward toward products ore back ward toward reactants. In effect, achievement of the transition state places a reacting system in a poised and precariously balanced position at the top of the activation energy barrier.

For example, in the transfer of a phosphate group from one molecule to another, a transition state is set up in which both molecules (shown as X and Y in Figure 2) link to the phosphate group a fraction of a second via transitory bonds (dotted lines). This unstable state can change readily in the direction of either products or unchanged reactants.

A number of mechanisms operate to contribute to formation of the transition state. One is bringing reacting molecules into close proximity. Many reactions involve combination or interaction of two or more reactant molecules. For the reaction to take place, the substrate molecule must collide. The required collisions may be rate among reactant molecules suspended in free solution, particularly if the substrates are present in low concentrations. Binding at the active site of an enzyme brings the reactants close together, raising their effective concentration in the active site to many time the concentration in the surrounding solution.

A second contribution mechanism is orienting reactants in positions favoring their interaction. Binding at the active site may bring substrate molecules into an arrangement in which they can collide ate exactly the correct positions and angles required for achievement of the transition state.

The third contributing mechanism is exposing reactant molecules to altered environments that promote their interaction. Some reactions, for example, take place more readily in nonpolar environments. Active sites may create such an environment by binding reactants so closely that water molecules are excluded. Another important environmental change is creation of acidic or basic conditions by groups in the active site that release or take up H+ .

Factors Affecting Enzyme Activity

A number of external factors affect the activity of enzymes in speeding conversion of reactants to products. These factors, including variations in the concentration for substrate molecules, temperature, and pH, speed or slow enzymatic activity in highly characteristic patterns.

Substrate Concentration

Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, there reaction rate initially increases proportionately as collisions between enzyme molecules and reactants become more frequent Figure 3. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At the point at which the enzymes are cycling as rapidly as possible, further increases in substrate concentration have no effect on there reaction rate. At this point the enzyme is saturated and the reaction remains at the saturation level.

If the reaction reaches a point at which further increases in reactants have no effect in increasing the rate of the reaction, then there is a good chance that the reaction is catalyzed by an enzyme. Uncatalyzed reactions, in contrast, increase the rate rate of the reaction almost indefinitely as the concentration of reactants increases.

Temperature

A higher temperature generally results in an increase in enzyme activity. As the temperature rises, the movement of enzyme molecules and substrate molecules increases. This causes more collisions between enzyme and substrate and the net result is the formation of more product. If the temperature rises beyond a certain point, however, the enzyme activity eventually levels out and then declines rapidly because the enzyme is denatured by heat. The enzyme shape change during denaturation and can not catalyze the reaction and there is shape drop in the rate of the reaction with change in temperature.

The effect of pH on Enzyme Activity

A change in pH can also effect enzyme activity. Each enzyme has an optimal pH range that help maintain its normal configuration in an environment which it operates. Pepsins is a proteolytic enzyme found in the stomach and functions at pH 2. At pH 2 the tertiary structure of pepsin is not altered and will catalyze the reaction. Trypsin is and enzyme found in the small intestine and prefer a pH of about 8. The tertiary structure of a protein depends on interactions such as hydrogen bonding, between R groups. A change in pH can alter the ionization of these side chains and disrupt the normal configuration and in some case denature the enzyme. A denatured protein can not combine with a substrate.

Enzyme Inhibition

The fact that enzymes combine briefly with their reactants makes them susceptible to inhibition by unreactive molecules that resemble the substrate. The inhibiting molecules can combine with the active site of the enzyme but tend to remain bound without change, blocking access by the normal substrate. As a result, the rate of there reaction slows. If the concentration of the inhibitor becomes high enough, the reaction may stop completely. Inhibition of this type is called competitive because the inhibitor competes with the normal substrate for binding to the active site.

Some inhibitors interfere with enzyme-catalyzed reactions by combining with enzymes at locations outside the active site. These inhibitors, rather than reducing accessibility of the active site to the substrate, cause changes in folding conformation that reduce the ability of the enzyme to lower the activation energy. Because such inhibitors do no directly compete for binding to the active site, their pattern of inhibition is called noncompetitive. Some poisons or toxins exert their damaging effects by acting as enzyme inhibitors. For example, the action of cyanide and carbon monoxide as poisons depends on their ability to inhibit enzyme important the utilization of oxygen in cellular respiration. Poisons and toxins typically act irreversibly by combining so strongly with enzymes, either covalently or nocovalently, that the inhibition is essentially permanent. Some irreversible poisons, rather than combining with the enzyme, destroy enzyme activity by chemically modifying critical amino acid side groups.

The cell has built in mechanisms to control directly both enzyme concentration and activity. First cells are able to regulate whether an enzyme is present at all. This type of control regulates protein synthesis and will be discussed in a later chapter. Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.

In noncompetitive inhibition, a molecule binds to an enzyme but not at the active site. The other binding site is called the allosteric site (allo - other and steric -structure or space). The molecule that binds to the allosteric site is an inhibitor because it causes a change in the 3-dimensional structure of the enzyme that prevents the substrate from binding to the active site. In cells inhibition usually reversible; that is the inhibitor isn't permanently bound to the enzyme. Irreversible inhibition of enzymes also occurs, due to the presence of a poison. For example, penicillin cause the death of bacteria due to irreversible inhibition of an enzyme needed to form the bacterial cell wall. In humans, hydrogen cyanide irreversibly bind to a very important enzyme (cytochrome oxidase) present in all cells, and this accounts for its lethal effect on the body.

The activity of almost every enzyme is a cell is regulated by feedback inhibition. Feedback inhibition is an example of common biological control mechanism called negative feedback. Just as high temperature will cause furnace to shut off, in a similar manner the product of an enzyme can inhibit a enzyme reaction. When the product is in abundance, it binds competitively with its enzyme's active site; as the product is used up, inhibition is reduced and more product can be produced. In this way the concentration of the product is always controlled within a certain range.

Most enzymatic pathways are also regulated by feedback inhibition, but in these cases the end product of the pathway binds at an allosteric site on the first enzyme of the pathway. This binding shuts down the pathway, and not more product id produced. The reaction series converting theronine to isoleucine is a classic example of allosteric regulation. Five enzymes acting in sequence catalyze the pathway. The final product of the sequence, isoleucine, acts as an inhibitor of the first enzyme of the pathway, threonine deaminase. As the pathway produces isoleucine, any molecules made in excess of cell requirements combine reversibly with threonine deaminase at a location outside the active site. The combination converts threonine deaminase to the T state and inhibits its ability to combine with threonine. The pathway is then turned off. If the concentration of isoleucine later falls as a result of its use in cell synthesis, isoleucine releases from the threonine deaminase enzymes, converting them to the R state in which they have high affinity of the substrate, conversion of threonine to isoleucine takes place.

Activation of an allosteric enzyme by an activator is another form of feedback inhibition. Combination of the activator and the allosteric site cause a conformational change in the active site permitting substrate binding and the reaction will be caltalyzed.