Biology - Topic 2: enzymes

What are enzymes? (not function)

complex, 3D, gobular proteins with a specific tertiary shape

What bonds form the overall 3D shape of an enzyme?

> hydrogen

> ionic

> disulfide bridges

> hyrophilic and hydrophobic

Metabolism?

the chemical reactions of an organism

Two types of metabolic reactions?

breaking down and building up

Anabolic reaction?

larger molecules are built up from smaller ones

Examples of an anabolic reaction?

the synthesis of proteins

Catabolic reaction?

larger molecules are broken down into smaller ones

Example of a catabolic reaction?

the digestion of starch into sugar by amylase

How do enzymes work?

> The substrate fits into the enzyme's active site which forms the enzyme-substrate complex

> Bonds form between some of the amino acids in the enzyme and substrate

> The substrate becomes an enzyme-product complex so that the products no longer fit in the active site and are released

Activation energy?

The energy required to initially break bonds

How do enzymes speed up the rate of reaction?

They lower the activation energy

Graph for enzyme energy barrier

page 5

What is the active site?

the part of the enzyme molecule to which the substrate binds

How does the active site work?

> The active site is where certain amino R-groups are exposed

> it is complementary to the substrate

> the substrate fits into the active site and interacts with these R-groups of amino acids by ionic and hydroge bonding

> this forms the enzyme substrate complex

Enzyme specifity?

one type of substrate can be catalysed by only one type of enzyme

Lock and key model?

the shape of the enzyme's active site is complementary to the shape of the substrate. They fit together like a lock and key

Induced fit model?

The active site of the enzyme very closely matches the shape of the substrate but can mould itself around the substrate, forming a precise fit

> the active site is flexible

What are cofactors?

non-protein substances that some enzymes require in order to function

Examples of cofactors?(3)

> metal ions

> prosthetic groups

> coenzymes

How do metal ions act as cofactors?

they form attachments to the enzyme changing the shape of the active site, enabling a reaction to take place

Examples of metal ion cofactors?

Mg2+, Ca2+, Fe3+

Example of a prosthetic group?

Haem in catalase

What are coenzymes?

another type of cofactor which are non-protein organic molecules that aren't permanently attached

Examples of coenzymes?

> NAD

> FAD

What factors effect the activity of enzymes?(5)

> Temperature

> pH

> concentration of enzyme

> concentration of substrate

> presence of inhibitors

Graph for the effect of temperature on enzymes?

Description of enzyme and temperature graph?

> As temperature increases, the rate of reaction increases

> once the optimum temperature is exceeded the rate of reaction decreases rapidly

Explanation of enzyme and temperature graph?

> As temperature increases the molecules gain more kinetic energy and therefor more faster

> There are more successful collisions and more enzyme-substrate complexes are formed

> more products will be made

> once the optimum is exceeded the high temps break the hydrogen bonds in the tertiary structure of the enzyme which alters the shape of the active site and stops substrates from entering and being broken down

Graph of effect of pH on enzyme activity?

Description of the effect of pH on enzyme activity?

> As the pH increases the rate of reaction increases until the optimum pH is reached

> once the optimum pH is exceeded the rate of reaction will decrease at the same rate

Explanation for the effect of pH on enzyme activity?

> As the pH deviates from the optimum, the ionic bonds within the tertiary structure are broken which alters the shape of the enzyme's active site and doesn't allow substrates to fit and be broken down

> less products will form

Graph for the effect of enzyme concentration on enzyme activity?

page 19

Description of the graph for the effect of enzyme conc on rate of reaction?

> As the enzyme conc increases, so does the rate of reaction

> the graph eventually levels off

Explanation for the effect of enzyme conc on rate of reaction?

> At low enzyme conc there is great competition for the active sites so rate of reaction is low

> As the enzyme conc increases more enzyme active sites become available to form enzyme-substrate complexes

> more reactions can take place and more products will form

> the graph levels off when the substrate molecules become the limiting reactant

Graph for the effect of substrate concentration on rate of reaction?

Description of graph for the effect of substrate concentration on rate of reaction?

> As substrate con increases, the rate of reaction increases

> the graph eventually levels off

Explanation for the graph for the effect of substrate concentration on rate of reaction?

> At low substrate conc the rate of reaction is low because the substrates occupying all the active sites they can but there are still some unoccupied

> substrate conc is the limiting reactant

> the graph eventually levels off because all of the enzyme's active sites are in use

> enzyme conc becomes the limitng reactant

Name for the point at which the graph for substrate conc levels off?

point of saturation

What are inhibitors?

substances that prevent enzymes from forming enzyme-substrate complexes and so stop or slow down the catalysis of the reaction

How does the inhibitor affect the rate of reaction?

> the substrate can't bind to the active site

> no enzyme substrate complexes can form

> so no product is formed

> rate of reaction decreases or is zero

Two types of inhibitors?

> competitive

> non-competitive

How do competitive inhibitors work?

> molecules that are complementary to all or part of the active site and compete with the substrate for the active site

> blocks the active site so substrate can't enter and form enzyme-substrate complex

Graph for the effect of a competitive inhibitor on substrate concentration?

How to reduce the effect of a competitive inhibitor?

increase substrate concentration

How does a non-competitive inhibitor work?

the inhibitor binds to the enzyme at the allosteric site and changes the shape of the active site or blocks in irreversibly so the substrate is no longer complementary

Effect of increasing substrate concentration when non-competitive inhibitors are present?

increase in substrate concentration doesn't decrease the effect of the inhibitors

Graph for the effect of non-competitive inhibitors on substrate concentration?

Are inhibitors reversible?

many inhibitors are reversible but some permanently damage the active site by breaking the bonds in the tertiary structure

How are enzymes used as biomarkers of disease?

Some diseases cause the presence of enzymes so they can be used as diagnostic tools or to track the progression of a disease

Where will these enzymes caused by diseases be found?

> blood

> urine

> sputum

Example of an enzyme produced because of illness?

Elastase

When is elastase produced?

Released by white blood cells due to lung infection

Advantage of elastase?

Elastase breaks down bacterial pathogens

Disadvantage of elastase?

Breaks down the protein elastin which contributes to the elasticity of the alveoli

Inhibitor for elastase?

Alpha-1-antitrypsin (A1AT) which prevents elastase activity after the infection has been cleared

How can cigarette smoke effect elastase?

> cigarette smoke increases production of elastase

> smoke inhibits production of A1AT

> elastin in alveoli is broken down

Lung disease cause by elastase?

elastase-induced emphysema

How can enzyme inhibitors work as therapeutic drugs?

enzyme inhibitors can target enzymes which are causing diseases so the progression of the disease can be slowed

Requirements for enzyme inhibitors to be effective?

> specific so only works on target enzyme

> works at low doses so no inhibitor build up as toxic

Examples of inhibitors used as therapeutic drugs?(4)

> A1AT - reduces harmful effects of lung infection

> ACE - treat high BP by stopping enzymes that constrict the blood vessels

> Penicillin - inhibits enzymes that create bacterial cell walls

> Anti viral drugs - inhibit DNA/RNA polymerase

Current research on inhibitors as therapeutic drugs?

trying to work out shape of active site of certain disease causing enzymes so complementary inhibitors can be created

How to maximise efficiency of enzymes?

Immobilise them

What is immobilization of enzymes?

Attaching enzymes or putting inside an insoluble support

Methods of immobilization?(4)

> Adsorption

> Cross linkage

> Entrapment

> Encapsulation

Adsorption?

the enzymes are attached by weak forces to an inert substance such as glass or a matrix

Cross linkage?

The enzymes are bonded covalently to a matrix, such a cellulose, as a consequence of chemical reactions

Entrapment?

the enzymes are trapped within polymers such as alginate beads or microspheres

Encapsulation?

The enzymes are trapped inside a selectively permeable membrane

Advantages of immobilising enzymes?(5)

> thermostable

> more resistant to wider pH range

> enzymes can be retained and reused: cheaper

> product is enzyme free which simplifies downstreaming, reduces purification costs and avoids possible allergic reaction

> commercial processes can be continuous: faster + less waste

Disadvantage of immobilising enzymes?

enzyme activity reduced bcs not all active sites are available and it takes time for substrate to diffuse

Why are enzymes very effective biosensors?

> specific

> quantitative

How do biosensors work?

molecule being monitored attaches to immobilized enzymes in reaction causing colour change or is converted to an electrical signal

Example of biosensors?

> clinistix

> digital blood glucose monitors

How to pregnancy tests work?

the antibody in the test strip reacts with specific proteins(hormones) to give a colour change due to antibody-enzyme complexes forming

How are enzyme inhibitors used in diagnostic reagent strips?

inhibitors are attached to the strip and if the enzyme attaches to the inhibitor a colour change will occur

> currently used to test for early cardiovascuclar disease and pre-eclampsia