ENZYMES: How Enzymes Work, Factors Affecting Enzymes (1), Factors Affecting Enzymes (2), Classification of Enzymes, Enzyme Technology

HOW DO ENZYMES WORK

Metabolic reactions take place in a number of steps, with each intermediate step forming an intermediate molecule on the way to the final product. The intermediate molecules are less stable (contain more energy) than the reactants or products. This is called the transition state.

Enzymes are biological catalysts that:

- increase in great amounts the rate of chemical reactions whitout the enzymes being changed in the reactions.
- they can be reused (small concentrations are very effective).
- they do not alter the final amount of product formed, just the speed at which it is formed.

The diferences between inorganic catalists (i. c.) and enzymes (e.) are the following:

- i. c. catalyse many different types of reactions, while e. catalyse only one type of chemical reaction.
- i. c. are not very affected by T and pH, while e. are affected by T and pH.
- i. c. work effectively in widely varying conditions of T and pressure, and extremes of pH, while e. work effectively only in a limited range of T, pH and pressure.

Lock and Key Theory:

· The substrate fits into a rigid active site.
· Various types of bonds (including hydrogen and ionic bonds) hold the substrate in the active site to form an enzyme-substrate complex.
· Once this is formed, enzymes change the substrate (either splitting it apart or linking pieces together).
This theory explain why enzymes are specific and why any change in the enzyme shape, no matter how small, alter its effectiveness (the shape of the substrate must fit in the enzyme exactly if a reaction is to be catalyzed).
However, this theory isn’t a totally satisfactory explanation for enzyme action, because this would depend on the unlikely event of randomly moving substrate molecules entering to the active site.

Induced-Fit Theory:

· The active site is able to change its shape to enfold a substrate molecule.
· The enzyme takes up its most effective catalytic shape after binding whit the substrate.
· The distorted enzyme molecule in turn distorts the substrate molecule, straining or twisting the bonds.
· This makes the substrate less stable, reduces its potential energy, and lowers the activation energy of the reaction.
· The reaction occurs and products are formed whit no longer bind to the active site, so it moves away.
· The flexible enzyme returns to its original shape ready to bind the next substrate molecule.



FACTORS AFFECTING ENZYMES (1)

Substrate concentration:

+ rate of an enzyme-catalysed reaction / + substrate concentration

The reaction will increase unitl it reaches a maximun rate. After this, all the active sites of the enzymes will be filled, so more substrate concentrarion will have no effect on the rate

of the reaction.

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 (∆A / ∆T). This is represented graphically in this Figure.

Enzyme concentration:

+ enzyme concentration / + rate of enzyme-catalysed reaction

This will happen as long as no other factors are limitng the rate. pH, pressure, and T° are constant, and substrates are present in excess concentrations.

NOTE: the initial rate of an enzyme-catalysed reaction is need to compare the rate under different conditions. The rate of reaction is usually expressed as the amount of substrate converted into product per unit of time. (We suggest you to check figure 2, on page 44 of the book).

Incubation time:

+ incubation time / - rate of an enzyme-catalysed reaction

The incubation time is the lenght of time over which a reaction has taken place. While the incubation time gets higher, the rate decreases because the enzyme gradually becomes denatured with time (as the protein molecule deforms, it loses it effectiveness as an enzyme).

Temperature:

+ rate of an enzyme-catalysed reaction / T° increases until the optimum temperature.

At sub-optimal T°, increasing T° increases the kinetic energy of the substrates (as they move faster, they are more likely to collide and interact with each other and with the enzyme).
Temperature coeficient (Q10): the change in the rate of reaction for each 10°C rise in T°:

Q10 = rate of reaction at x + 10°
rate of reaction at x°C

Above the optimum temperature, the rate falls because the increased energy cause bonds that maintain the enzyme shape to break, and the enzyme becomes denatured.

Hydrogen Ion concentration (pH):

Enzymes are effective in only a narrow pH range, an within this range, there is an optimum pH at wich activity is the greatest (the optimum pH usually matches the usual pH environment of the enzyme).
Deviations from the optimum pH can cause bonds to be broken (especially hydrogen and ionic bonds), so that the enzymes becomes denatured.

FACTORS AFFECTING ENZYMES (2)

Cofactor: is a non-protein susbtance. A cofactor that is tightly bond to its enzyme is called a prostethic group. There are two types of cofactors:
Inorganic – Activators: they attach to the active site of the enzyme to make its shape more efficient (e.g.: copper, iron or zinc).

Organic – Coenzymes: they transfer chemical groups, atoms or electrons from one enzyme to another (e.g.: vitamins).

Inhibitors: they are substances that can interfere with enzymes, reducing or even completely destroying their action. There are two types of inhibitors*:
Competitive:
- They have a shape resembling the enzyme’s normal substrate.
- They compete with the substrate to occupy the active site (if the inhibitor occupies it, it prevents the enzyme from combining with its normal substrate).
- The effect of the competitive inhibitor depends on its concentration compared to the substrate’s and on how tightly the enzyme binds to the inhibitor and substrate.

Non Competitive:
- They don’t attach to the active site but binds with the enzyme at another site (the allosteric site).
- Once attached, the non-competitive inhibitor causes the active site to change shape, preventing the normal substrate from binding here.
- The end product of a reaction can act as a non-competitive inhibitor, controlling series of enzyme-catallysed reactions. This is called end-product inhibition.

*Either type of inhibitors can be reversible or irreversible:
Reversible inhibitors: bind to an enzyme with weak bonds, such as hydrogen bonds which are easily broken. They affect the enzyme as long as they are attached to it (when they are detached, the enzyme functions normally again).

Irreversible inhibitors: attach to an enzyme with strong covalent bonds which are difficult to break without damaging the enzyme. Consequently, the effect of an irreversible inhibitor is permanent.

Metabolic pathway: the steps of metabolic reactions; regulated by the final susbtrate produced by it.

1) When the final substance produced by it reaches a certain concentration, it acts as a non-competitive inhibitor.
2) It binds onto the enzymes in the matabolic pathway (often the first one) and prevent reaction series from progressing.
3) The reactions start up again when the concentration of the end product falls to a low level. As we said before, this is called end-product inhibition.

NOTES:
· The enzyme that is inhibited, which functions as a regulatory enzyme in the metabolic pathway, is an allosteric enzyme (has a site separate from the active site to which an other substance can bind).
· The binding substance may be either an inhibitor or an activator (so may be either slowing down or speeding up the reaction).

ATP production is regulated by end-product inhibition:

1) ATP is produced by cellullar respiration until the level of ATP is high.
2) At this high concentration, ATP acts as an allosteric inhibitor, stopping any further production.
3) When ATP concentration falls (e.g.: by contracting muscle cells) cellullar respiration is no longer inhibited and ATP production starts up again.

CLASIFICATION OF ENZYMES

Oxydoreductases: catalyse redox reactions by the transfer of H2, O2 or electrons from one molecule to another.

Ethanal + NADH + H+ -alcohol dehydrogenase-> ethanol + NAD+

Transferasas: catalyse the transfer of a group of atoms from one compound to another.

glutamic acid + pyruvic acid -amino transferase-> alpha – ketoglucaric acid + alanine

Hydrolases: catalyse the splitting of a large substrate molecule into two smaller compounds. Water is involved (hydrolisis).

Lactose + H20 -lactase-> glucose + galactose

Lysases: catalyse the addition of a group across a double bond.

Pyruvic acid -pyruvic decarboxylase-> ethanal + carbon dioxide

Isomerases: catalyse rearrangements within a molecule, converting one isomer to another.

Glucose 1 - phosphate -phosphoglucomutase-> Glucose 6 - phosphate

Lygases: catalyse bond formation between two compounds. The reaction uses energy that comes from the hydrolysis of ATP to ADP and phosphate.

Amino acid + specific RNA -aminoacyl – tRNA synthetase + ADP + P-> amino acid tRNA complex + ADP + P

Types of enzyme nomenclature:
- Trivial names – suffix “ase”.
- Systematic names.

ENZYME TECHNOLOGY

Enzyme technology studies industrial enzymes and their uses.
These enzymes work at room T°, atmospheric pressure, moderate pH ranges and are more specific than inorganic catalysts.

Ways of employing enzyme technology:

Producing the enzymes: culturing the microbes:
The microorganisms may have specific genes introduced into their DNA by genentic engineering, so that they produce enzymes naturally made by other organisms.

Isolating the enzymes:
To obtain an intracellular enzyme, the microbe cells are harvested from the culture and broken down. Then the enzyme is precipitated from the solution by a salt or an alcohol (the enzyme can be purified by techniques such as electrophoresis or column chromotography). Extracellular enzymes are soluble in water, so they can be extracted from the culture medium and purified.

Using whole cells:
The substrate has to diffuse into the cells before the reaction takes place. The product may diffuse out of cells, or the cells disrupted to release it. The product may be extracted and purified.

Improving the enzyme: enzyme stability:
The enzyme stability is their ability to retain the tertiary structure.
Industries need enzymes to work in conditions they cannot, so thermophilic bacteria produce thermostable enzymes that don’t denature at high T°, are resistant to organic solvents and tolerate a wide range of pH. So the gene for this enzyme has been isolated and transfered to Bacillus subtilis (the microbe used in industries).

Improving the enzyme: immobilisation:
Unstable enzymes may be immobilised by being attached to or located within an insoluble support (stability increased).
Immobilisation’s advantages:
· Enzymes can recover easy and be used over and over again.
· The reaction products are not contamined by the enzyme (because they are in an inert matrix).
· Enzymes can be easily manipulated.
· Enzymes make continous production of a substrate easier.

Advantages and disadvantages of different types of immobilisation:

ENERGY AND METABOLISM

What is energy and its types

Every living organism on earth requires energy to stay alive. Energy is the capacity to do work: the ability to move matter in a direction in which it would not move without an imput of energy. The amount of matter may be small or large. Energy exists in many forms, including chemical, electrical, nuclear, heat, light, and mechanical energy. There are two types of mechanical energy: kinetic and potential. The first one is when matter that is moving and performing work. When matter is not actually performing a work, but that has the ability to do so is called potential energy. Also exists chemical energy, is equal to potential energy but in the atoms

Laws of thermodinamics

All organisms survive by transforming energy into another. Energy that can be used to do useful work, is called free energy.

• The first law of thermodinamics: states that energy can be neither created nor destroyed; it can only be changed from oneform into another. Therefore, when energy changes take place within an organism, the energy imput always equals the energy output.
  
• The second law of thermodinamics: this one is related to the fact that when energy is transformed from one type to another some of the energy is converted into heat.

Chemical reactions and metabolism

This ones lead to a chemical change in matter. They can be represented by any chemical equation, for example: 2H2+O2 ---> 2H2O. The two melecules of hidrogen (2H2) and one molecule of oxigen (O2) are called reactants or substrates. The two molecules of water, (2H2O) are called products. All equations are balanced this way, reflecting the fact that chemical reactions do nor destroy or create matter.

Cellular metabolism is the sum of all the reactions that the cell carries out, there are two main types of metabolic reaction: catabolism and anabolism. During catabolism substances break down and release energy. During anabolism, chemical reactions take in energy to synthesise complex molecules from simple molecules. Reactions that liberate more energy than they take in are called exergonic; those that take in more energy than they liberate are called endergonic.

Activation energy

Most molecules are in a relatively stable and require an input of energy to react with each other.

There is an energy barrier to the reaction. The amount of energy required to overcome this barrier and start a reaction is called the activation energy.

Catabolic reactions are exergonic (give out energy) because they have a small activation energy compared with the energy released during the reaction: the reaction products contain less energy than the substrates.

Anabolic reactions are endergonic because their activation energy is greater than the energy released during the reaction. The products of the reaction contain more energy than the reactants, therefore extra energy must be supplied for the reaction to proceed.