Enzymes

REACTIONS AND ENZYMES

Endergonic and exergonic

Energy releasing processes, ones that "generate" energy, are termed exergonic reactions.

Reactions that require energy to initiate the reaction are known as endergonic reactions.

thermodynamics

All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases.

endergonic reaction

Catabolism and Anabolism

Anabolism is the total series of chemical reactions involved in synthesis of organic compounds.

Autotrophs must be able to manufacture (synthesize) all the organic compounds they need.

Heterotrophs can obtain some of their compounds in their diet (along with their energy). For example humans can synthesize 12 of the 20 amino acids, we must obtain the other 8 in our diet.

Catabolism is the series of chemical reactions that breakdown larger molecules. Energy is released this way, some of it can be utilized for anabolism. Products of catabolism can be reassembled by anabolic processes into new anabolic molecules.

Enzymes: Organic Catalysts

Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system.

Enzymes function as organic catalysts.

A catalyst is a chemical involved in, but not changed by, a chemical reaction.

Many enzymes function by lowering the activation energy of reactions.

By bringing the reactants closer together, chemical bonds may be weakened and

reactions will proceed faster than without the catalyst.

The use of enzymes can lower the activation energy of a reaction (Ea).

Enzymes can act rapidlyAs in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood.

There are over 2000 known enzymes, each of which is involved with one specific chemical reaction.

Enzymes are substrate specific.

The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth).

Enzymes are proteins.

The functioning of the enzyme is determined by the shape of the protein.

The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will "fit".

It recognizes, confines and orients the substrate in a particular direction.

Space filling model of an enzyme working on glucose. Note the shape change in the enzyme (indicated by the red arrows) after glucose has fit into the binding or active site.

The induced fit hypothesis suggests that the binding of the substrate to the enzyme alters the structure of the enzyme, placing some strain on the substrate and further facilitating the reaction.

Cofactors are nonproteins essential for enzyme activity. Ions such as K+ and Ca+2 are cofactors.

Coenzymes are nonprotein organic molecules bound to enzymes near the active site. NAD (nicotinamide adenine dinucleotide).

Formation of an enzyme-substrate complex.

Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme.

The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process.

Also, intermediate products tend not to accumulate, making the process more efficient.

Equilibrium -the tendency of reactions to reverse when concentrations of the products build up to a certain level

By removing intermediates (and by inference end products) from the reactive pathway, equilibrium effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the "preferred" direction.

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 at which spontaneous reactions take place

Rate is equal to the number of reactant molecules converted to product per unit of time.

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.

Enzyme and Activation EnergyActivation energy is the energy barrier over which the molecules in a system must be raised for a reaction to take place.

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.

Shown to the right are two plots of the energy levels of molecules involved in a reaction such as

A + B > C

over the course of the reaction.

Activation Energy and Enzymes

The blue curve depicts the course of the reaction in the absence of an enzyme which facilitates the reaction while the red curve depicts the course of the reaction in the presence of the reaction specific enzyme.

The difference in energy level between the beginning state (left side) and the energy necessary to start the reaction (peaks of the curves) is the activation energy of the reaction.

The presence of the enzyme lowers the energy of activation.

Activation Energy and Enzymes

Shown to the right are three plots of the energy levels of molecules involved in a reaction such as

A + B > C

The first plot depicts the distribution of energy levels of the reactants at body temperature without the presence of an enzyme.

The vertical blue line indicates the activation energy level which must be attained for the reaction to occur.

The shaded portion of the distribution on the left indicates the proportion of A and B molecules having sufficient energy levels to participate in the reaction.

The second plot depcits the distribution of energy levels of the reactants at a temperature higher than body temperature but in the absence of an enzyme.

The distribution is shifted to a higher average energy level but the activation energy remains the same. Clearly, there is a higher proportion of molecules at an energy level high enough to participate in the reaction than if the molecules were at body temperature.

The third plot is the same as the first one but with the presence of an enzyme the activation energy for the reaction has been lowered significantly.

Thus, the proportion of molecules at an energy level above the activation is greatly increased which means the reaction will proceed at a much higher rate.

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.

Characteristics of enzymes proteins

1. Enzymes combine briefly with reactants during an enzyme-catalyzed reaction.

2. Enzymes are released unchanged after catalyzing the conversion of reactants to Product

3. Enzymes are specific in their activity; each enzyme catalyzes the reaction of a single type of molecules or

a group of closely related molecules.

4. Enzymes are saturated by high substrate concentrations.

5. Many enzymes contain nonproteins groups called cofactors, which contribute to their activity.

Inorganic cofactors are all metallic ions.

Organic cofactors, called coenzymes, are complex groups derived from vitamins.

6. Many enzymes are pH and temperature sensitive

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.

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.

How Enzymes Lower the Energy of Activation

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 link to the phosphate group a fraction of a second via transitory bonds.

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 at 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+ .

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.

ADP

Glucose-6-phosphate

Factors Affecting Enzyme Activity

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.

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 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.

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 effect of pH on Enzyme Activity

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.

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.

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.

Enzyme

Subsrate

Allosteric inhibitor

Enzyme

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.

Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.

Feedback inhibition

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.

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 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 no more product is 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.

Allosteric Activator

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.

Su

bstr

ate

Enzyme

Allosteric Site

Enzyme Enzyme

Su

bstr

ate

Allosteric Activator

http://csep10.phys.utk.edu/krogh_instructorCD/biology/ch6/animations/mod06_2.swf

http://www.northland.cc.mn.us/biology/Biology1111/animations/enzyme.html

Characteristics of Enzymes

1. Enzymes allow reactions to proceed at rates which are compatible with the state of some chemical-physical

systems which we define as "living”

2. Proteins which lower the energy barrier of reactions - act as "catalysts"

3. Influence rates only - do not affect the equilibrium state

4. Small amounts present - concentration dependency

Form reversible complex with substrate

5. Not consumed in the reaction - used over and over

6. Show specificity for a reaction - varies some for different enzymes

7. Fooled by inhibitors - forms the basis for some forms of chemotherapy

8. Each enzyme shows optimal conditions for activity - temperature, pH, etc.

9. Many enzymes require co-factors such as vitamins

10. 2,000 to 3,000 different enzymes in each cell - different cell types have different enzyme sets

11. Often involved in multi-step biochemical pathways

12. Subject to regulation by feedback control and other mechanisms

13. Production directly controlled by genes

14. Many "genetic diseases" are the result of faulty enzyme systems