F.6 Bio Notes 09-10 Cytology III Enzyme P.1

F.6 Bio notes_09-10 Cytology III Enzyme P.1

CYTOLOGY III : Enzymes

F.6 Bio notes_09-10 Cytology III Enzyme P.1

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F.6 Bio notes_09-10 Cytology III Enzyme P.1

nzymes are pro____. Each cell contains several hundred enzymes. They are biological cat_____. They speed up reactions. In their absence reactions in the cell would be too s___ to sustain life.

Enzyme reactions may be either ana____ (involved in synthesis) or cata____ (involved in breakdown). The sum total of all these reactions in a living cell or organism constitutes its metabolism.

Commonly, a number of enzymes are used in seq_____ to convert one substance into one or several products via a ser__ of intermediate compounds. The chain of reactions is referred to as a metabolic pathway. Thus enzymes serve to control the chemical reactions that occur within cells and ensure that they proceed at an efficientrate.

I)Properties of enzyme

• all are glo_____ proteins;
• they increase the rate of a reaction without themselves being u____ up;
• their presence does not alter the n_____ or properties of the end product(s) of the reaction;
• a very s____ amount of catalyst effects the change of a large amount of substrate;
• their activity varies with p__, t______, sub______and en______concentrations;
• the catalysed reaction is re______, both forward and backward reactions are catalysed; the equ______position of the reaction is not changed.
• they are sp_____, that is an enzyme will generally catalyse only a single reaction.

II)Catalysis and Activation Energy

The energy required to make substrates react is called act______energy [Ea].
The greater the amount of activation energy required, the slower the rate of reaction at a given temperature.
Enzymes serve to re____ the activation energy required for a chemical reaction to take place. /
A Possible mode of action:
An enzyme combines with its substrate to form a short-lived e_____-s______complex.
Within this complex the cha_____ of reactions occurring are greatly enhanced.
Once a reaction has occurred, the complex breaks up into p______and enzyme.
The enzyme remains un______ at the end of the reaction and is fr__ to interact again with more substrate. /

substrate + enzyme enzyme/substrate enzyme/product(s) enzyme + product(s)

complex complex

III)Specificity of Enzyme Action

Enzymes possess particular sh_____ (active site) into which the substrate or substrates fit exactly.
The 'lock and key' hypothesis:
Substrate is the key whose shape is comp______to the enzyme -- the lock.
The'induced-fit' hypothesis. /

In 1959 Koshland suggested a modification to the 'lock and key' analogy. He envisaged a dynamic inter______ occurring between enzyme and substrate. He argued that when a substrate combines with an enzyme, it induces ch____ in the enzyme structure. The amino acids which constitute the active site are mouldedinto a precise formation which enables the enzyme to perform its catalytic function most effectively. A suitable analogy would be that of a hand changing the shape of a glove as the glove is put on.

IV)Enzyme Cofactors

Many enzymes require non-pro____ components called cofactors for their efficient activity.

a)Inorganic ions

These are thought to m_____ either the enzyme or the substrate into a s_____ such that an enzyme/substrate complex can be formed. E.g.Salivary a______ activity is increased in the presence of chloride ions.

b)Organic cofactor- for example, haem

The organic molecule is integ____within the protein molecule in such a way that it effectively assists the catalytic function of its enzyme.

For example, H____ is an iron-containing organic complex. It has a number of biologically important functions:

i)Electron carrier. Haem is the prosthetic group of cytochromes (see respiratory chain), where it acts as an e_____ carrier. In accepting electrons the iron is reduced to Fe(II); in handing on electrons it is oxidised to Fe(III). In other words it takes part in ox_____/re______reactions by reversible changes in the valency of the iron.

ii)Oxygen carrier. Haemoglobin and myoglobin are o____-carrying proteins that contain haem groups, Here the iron remains in the reduced, Fe(II) form.

c)Coenzymes (for example NAD, NADP, ATP)

Coenzymes are small, non-protein molecules loo____ bound with the enzyme. They act as carr____ of atoms or groups from one place to another in the metabolic reaction. For example: Nicotinamide adenine dinucleotide (NAD).

NAD is derived from the vitamin nicotinic acid (Vitamin B ) and can exist in both a re____ and an ox_____ form. In the oxidised state it functions in catalysis as a hydrogen accepter:

AHNAD BH
A NADH B
E1 E2 /

(where E1 and E2are two different dehydrogenase enzymes. Net effect: H transferred from A to B. Here the coenzyme NAD acts as a link between two different enzyme systems E1 and E2.)

V)Factors affecting the rate of enzyme reactions

When investigating the effect of a given factor on the rate of an enzyme-controlled reaction, all other factors should be kept constant and at optimum levels wherever possible, initial rates only should be measured. Why?

a)Enzyme concentration

The rate of reaction is pro______to the enzyme concentration.
-- provided that the substrate concentration is not lim______, and other conditions such as pH and temperature are opt______, /

b) Substrate Concentration

F.6 Bio notes_09-10 Cytology III Enzyme P.1

For a given enzyme concentration, the rate of an enzymatic reaction increases with increasing s______concentration.
There comes a point when any further increase in substrate concentration produces no significant change in reaction rate.
This is because at high substrate concentrations the a_____ sites of the enzyme molecules at any given moment are virtually sat______with substrate. /

c)Temperature

The effect of temperature on the rate of a reaction can be expressed as the temperature coefficient, Q10

rate of reaction at (x + 10) ° C

Q10

rate of reaction at x ° C

Over a certain range oftemperature,Q10 for an enzyme reaction is 2. i.e. the rate of an enzyme reaction d______for every rise of 10 ° C.
Heat increasesk_____ energy and hence molecular motion, thus the reactants move more quickly and chances of their bumping into each other with sufficient energy to react are increased.
The temperature that promotes maximum activity is referred to as the op______temperature.
If the temperature is increased above this level, then a decrease in the rate of the reaction occurs despite the increasing frequency of col______.
This is because the secondary and t______structures of the enzyme have been disr______, and the enzyme is said to be de______. /

If temperature is reduced to near or below freezing point, enzymes are inact_____, NOT denatured. They will res___ their catalytic influence when higher temperatures are restored.

/ Quick-freezing food are in widespread use as a means of preserving food for extensive periods.
This not only prevents growth and multiplication of micro______,
but also inactivates the nat_____ enzymesin the foods itself..
Are all enzymes denatured at high temperatures? Look up for thermophilic bacteria and be ready to explain how they survive in near boiling waters.

d)pH

Most enzymes function only in a n_____ pH range. When the pH is above or below this value, the rate of enzyme activity diminishes. Changes in pH alter the ionic ch_____ of the acidic and basic groups that help to maintain the specific s_____ of the enzyme. WHY?

The pH change leads to an alteration in enzyme shape, particularly at its active site. If extremes of pH are encountered by an enzyme, then it will be de______.

F.6 Bio notes_09-10 Cytology III Enzyme P.1

VI) Enzyme inhibition

Enzyme inhibitors are molecules that b____ to enzymes and dec_____ their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many dr___ are enzyme inhibitors. They are also used as pest_____.

a)Reversible Inhibition

Reversible inhibitors bind to enzymes with relatively w____, non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds.

In contrast to irreversible inhibitors, reversible inhibitors generally do NOT undergo che_____ reactions when bound to the enzyme and can be easily removed by dil____ or dialysis.

i)Competitive reversible inhibition

Here a compound, stru______similar to that of the usual substrate, associates with the enzyme's active site, but is unable to react with it. While it remains there it prevents access of any molecules of true substrate. The genuine substrate and inhibitor com____ for position in the active site.

It is able to be rev_____, for if the substrate concentration is increased, the rate of reaction will be inc______.i.e., by out-competing the inhibitor.

The knowledge of competitive inhibition has been utilised in chemotherapy. This is the use of chemicals to destroy infectious micro-organisms without damaging host tissues.

During the Second World War, sulphonamides, chemical derivatives of sulphanilamide, were used extensively to prevent the spread of microbial infection. The sul______are similar in structure to para-aminobenzoate (PAB), a substance essential to the growth of many pathogenic bacteria. The bacteria require PAB for the production of folic acid, an important enzyme cofactor. Sulphonamides act by interfering with the synthesis of folic acid from PAB.

Animal cells are insensitive to sulphonamides even though they require folic acid for some reactions. This is because they use pre-formed folic acid and do not possess the necessary metabolic pathway for making it.

ii)Non-competitive reversible inhibition

This type of inhibitor is NOTstructurally sim______ to the substrate. Theybind with the enzyme at a point other than its active site. (an allosteric site).

This affects the rate of the reaction catalysed by the enzyme because the presence of the inhibitor causes a change in the stru______and sh____of the enzyme. This change in shape means the enzyme is NO longer able to bind with a sub_____ correctly. This reduces the concentration of 'active' enzyme resulting in a decrease in the maximum rate of the reaction.

In this mode of inhibition, there is no com______ between the inhibitor and the substrate, so increasing the concentration of the substrate does NOT allow the maximum enzyme activity rate to be reached. As a result, the extent of inhibition depends only on the con______of the in______.

For example, cyanide combines with metallic ions (acting as prosthetic groups) of some enzymes (such as copper ions of cytochrome oxidase) and inhibits their activity. The rate of reaction will continue to decrease with increasing inhibitor concentration. When inhibitor saturation is reached, the rate of the reaction will be almost nil.

A comparison of competitive and non-competitive inhibition

competitive inhibition / non-competitive inhibition

1. substrate and inhibitor binding to the enzyme at the same time

1. inhibitors are often similar in structure to the real substrate / inhibitor having an affinity for the active site of an enzyme

1. the substrate and inhibitor compete for access to the enzyme's active site.

1. results from an allosteric effect where the inhibitor binds to a different site on an enzyme.

1. can be overcome by sufficiently high concentrations of substrate

1. Inhibition can be reduced, but not overcome by increasing concentrations of substrate. The extent of inhibition depends only on the concentration of the inhibitor

b)Irreversible inhibition

Very small concentrations of chemical reagents such as the heavy metal ions mercury (Hg2+), silver (Ag+) and arsenic (As+), completely inhibit some enzymes. They combine permanently with -sulphydryl (-SH) groups and cause the protein of the enzyme molecule to precipitate. If these are components of the active site then the enzyme is inhibited.

Q.What would be the effect on the rate of reaction between an inhibitor of this kind and the substrate if substrate concentration is increased?

c) End-product inhibition (negative feed-back inhibition)

When the end product of a series of metabolic pathway begins to accumulate, it may act as an inhibitor on the enzyme controlling the ini____ steps of the pathway.

The affinity of the enzyme for its substrate would therefore be lowered, and further production of the end product decreased or prevented.

This is called end-product inhibition and is an example of a (negative feedback mechanism) serving to control an aspect of metabolic activity.

F.6 Bio notes_09-10 Cytology III Enzyme P.1

1. What is the significance of End-product inhibition in the efficiency of metabolism ?

Q.What is the difference between enzyme inhibition and enzyme inactivation? In drug design, which one is more common and why?

Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target.

For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids. In making drugs that works by inhibition, their action need to be specific, therefore specific enzyme inhibition is employed rather than enzyme inactivation like the use of destructive agents such as conc acids!

Reference reading: Putting enzymes to use

Many of the reactions catalysed by enzymes have commercial uses, the conversion of starch to sugar being just one example. These reactions can be made to happen without enzymes, for example by heating and/or the use of strong acids. Indeed in the past this has been the main approach used in industry. However, enzymes with their fast but gentle action provide a promising alternative.

There are three main advantages of using enzymes in industrial processes, and they are directly related to the properties of enzymes.

• They are specific in their action and are therefore less likely to produce unwanted by-products.
• They are biodegradable and therefore cause less environmental pollution.
• They work in mild conditions, i.e. low temperatures, neutral pH and normal atmospheric pressure, and are therefore energy-saving.

The main disadvantage of enzymes is that they are highly sensitive to changes in the physical and chemical conditions surrounding them. Thus they may be readily denatured by even a small increase in temperature and are highly susceptible to poisons and changes in pH. This means that the conditions in which they work must be stringently controlled. In particular the enzyme-substrate mixture must be uncontaminated with other substances that might affect the reaction, and the equipment must be scrupulously clean.

To be effective in a production process the enzyme molecules must be brought into maximum contact with the substrate molecules. This is achieved in one of two ways.

• One way is simply to mix solutions of the enzyme and substrate in suitable concentrations.
• The other way is to attach the enzyme molecules to an inert surface such as plastic beads and then bring the surface into contact with a solution of the substrate.

This latter method has the advantage of enabling the enzyme molecules to be used over and over again, with the result that a lot of product can be made from a relatively small amount of the enzyme.

A generalised fermenter
/ Commercial enzymes are produced by micro-organisms such as yeasts and bacteria. Sometimes naturally occurring strains of these micro-organisms are used, but increasingly nowadays special strains are developed by genetic engineering to produce particular enzymes. The micro-organisms themselves are grown in vast fermenters from which their enzymes are removed as and when required.
Approximately 2000 enzymes have been identified, and of these over and letting in 150 are used in industrial processes. For example, amylases which convert medium starch to sugars are used for making syrups, fruit juices, chocolates and other food products.
Cellulases, which break down cellulose, are used for softening vegetables, removing the seed coat from cereal grain, and extracting agar jelly from seaweed. And proteases are used for tenderising meat, skinning fish and removing hair from hides. But perhaps the best known use of enzymes is in biological washing powders.

Biological washing Powders

A biological washing powder contains enzymes, usually proteases, which remove 'biological' stains such as food, blood and so on.

The advantage of modern biological washing powders is that they are effective at relatively low temperatures, and are therefore energy saving as well as gentler on the clothes. However, there are possible health hazardsthat need to be considered and investigated. After all, protease enzymes break down proteins under natural conditions inside the body. There may be unknown dangers in allowing such proteins to come into contact with the skin and mucous membranes.

The enzyme is produced by bacteria in a fermenter. The bacterial cells are separated from the culture medium by centrifugation. Any bacterial cells still present are filtered out. The enzyme is then separated from the liquid by precipitation with, for example, a salt. Next the enzyme is granulated with common salt which acts as a preservative, and mixed with water to form a paste. The paste is shaped into spheres. Finally the spheres are dried and coated with a layer of wax, then cooled. The finished particles are added to the other ingredients of the washing powder and packaged. The wax subsequently melts in the wash, releasing the enzyme. Why should the enzyme be coated with wax?

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