enzymes Flashcards
enzymes as catalysts
Enzymes are biological catalysts
‘Biological’ because they function in living systems
‘Catalysts’ because they speed up the rate of chemical reactions without being used up or undergoing permanent change
Enzymes are globular proteins with complex tertiary structures
Some are formed from a single polypeptide, whilst others are made up of two or more polypeptides and therefore have a quaternary structure
Metabolic pathways are controlled by enzymes in a biochemical cascade of reactions
Virtually every metabolic reaction within living organisms is catalysed by an enzyme
Enzymes are therefore essential for life to exist
site of action of enzymes
All enzymes are proteins that are produced via the process of protein synthesis inside cells
Some enzymes remain inside cells, whilst others are secreted to work outside of cells
Enzymes can therefore be intracellular or extracellular, referring to whether they are active inside or outside the cell respectively
Intracellular enzymes are produced and function inside the cell
Extracellular enzymes are secreted by cells and catalyse reactions outside cells (eg. digestive enzymes in the gut)
As well as amylase (which hydrolyses starch into maltose), another example of an extracellular digestive enzyme that is secreted by the pancreas and enters the small intestine is trypsin, which breaks proteins down into peptides and amino acids
In fact, some organisms can only feed using a form of extracellular digestion in which the digestive enzymes are actually secreted outside of their bodies
For example, the hyphae of fungi secrete the necessary enzymes directly onto the food they are consuming (e.g. wood) so that the food is digested into smaller, simple molecules that the fungi can then absorb through the walls of the hyphae
mechanism of enzyme action
Enzymes have an active site where specific substrates bind forming an enzyme-substrate complex
The active site of an enzyme has a specific shape to fit a specific substrate
Extremes of heat or pH can change the shape of the active site, preventing substrate binding – this is called denaturation (the enzyme is said to be denatured)
Substrates collide with the enzymes active site and this must happen at the correct orientation and speed in order for a reaction to occur
enzyme specificity
The specificity of an enzyme is a result of the complementary nature between the shape of the active site on the enzyme and its substrate(s)
The shape of the active site (and therefore the specificity of the enzyme) is determined by the complex tertiary structure of the protein that makes up the enzyme:
Proteins are formed from chains of amino acids held together by peptide bonds
The order of amino acids determines the shape of an enzyme
If the order is altered, the resulting three-dimensional shape changes
the enzyme-substrate complex
An enzyme-substrate complex forms when an enzyme and its substrate join together
The enzyme-substrate complex is only formed temporarily before the enzyme catalyses the reaction and the product(s) are released
the lock and key hypothesis
Enzymes are globular proteins
This means their shape (as well as the shape of the active site of an enzyme) is determined by the complex tertiary structure of the protein that makes up the enzyme and is therefore highly specific
In the 1890’s the first model of enzyme activity was described by Emil Fischer:
He suggested that both enzymes and substrates were rigid structures that locked into each other very precisely, much like a key going into a lock
This is known as the ‘lock-and-key hypothesis’
the induced fit hypothesis
The lock-and-key model was later modified and adapted to our current understanding of enzyme activity, permitted by advances in techniques in the molecular sciences
The modified model of enzyme activity (first proposed in 1959) is known as the ‘induced-fit hypothesis’
Although it is very similar to the lock and key hypothesis, in this model the enzyme and substrate interact with each other:
The enzyme and its active site (and sometimes the substrate) can change shape slightly as the substrate molecule enters the enzyme
These changes in shape are known as conformational changes
The conformational changes ensure an ideal binding arrangement between the enzyme and substrate is achieved
This maximises the ability of the enzyme to catalyse the reaction
enzymes and the lowering of activation energy
All chemical reactions are associated with energy changes
For a reaction to proceed there must be enough activation energy
Activation energy is the amount of energy needed by the substrate to become just unstable enough for a reaction to occur and for products to be formed
Enzymes speed up chemical reactions because they reduce the stability of bonds in the reactants
The destabilisation of bonds in the substrate makes it more reactive
Rather than lowering the overall energy change of the reaction, enzymes work by providing an alternative energy pathway with a lower activation energy
Without enzymes, extremely high temperatures or pressures would be needed to reach the activation energy for many biological reactions
Enzymes avoid the need for these extreme conditions (that would otherwise kill cells)
enzyme activity: pH
All enzymes have an optimum pH or a pH at which they operate best
Enzymes are denatured at extremes of pH
Hydrogen and ionic bonds hold the tertiary structure of the protein (ie. the enzyme) together
Below and above the optimum pH of an enzyme, solutions with an excess of H+ ions (acidic solutions) and OH- ions (alkaline solutions) can cause these bonds to break
The breaking of bonds alters the shape of the active site, which means enzyme-substrate complexes form less easily
Eventually, enzyme-substrate complexes can no longer form at all
At this point, complete denaturation of the enzyme has occurred
Where an enzyme functions can be an indicator of its optimal environment:
Eg. pepsin is found in the stomach, an acidic environment at pH 2 (due to the presence of hydrochloric acid in the stomach’s gastric juice)
Pepsin’s optimum pH, not surprisingly, is pH 2
buffer solutions used to measure the rate of reactions
When investigating the effect of pH on the rate of an enzyme-catalysed reaction, you can use buffer solutions to measure the rate of reaction at different pH values:
Buffer solutions each have a specific pH
Buffer solutions maintain this specific pH, even if the reaction taking place would otherwise cause the pH of the reaction mixture to change
A measured volume of the buffer solution is added to the reaction mixture
This same volume (of each buffer solution being used) should be added for each pH value that is being investigated
investigating the effect of pH on enzyme rates
Use the enzyme amylase to breakdown starch at a range of pH values, using iodine solution as an indicator for the reaction occurring
Amylase is an enzyme that digests starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)
A continuous sampling technique can monitor the progress of the reaction
Starch can be tested for using iodine solution
enzyme activity: temperature
Enzymes have a specific optimum temperature
This is the temperature at which they catalyse a reaction at the maximum rate
Lower temperatures either prevent reactions from proceeding or slow them down because:
Molecules move relatively slowly as they have less kinetic energy
Less kinetic energy results in a lower frequency of successful collisions between substrate molecules and the active sites of the enzymes which leads to less frequent enzyme-substrate complex formation
Substrates and enzymes also collide with less energy, making it less likely for bonds to be formed or broken (stopping the reaction from occurring)
Higher temperatures cause reactions to speed up because:
Molecules move more quickly as they have more kinetic energy
Increased kinetic energy results in a higher frequency of successful collisions between substrate molecules and the active sites of the enzymes which leads to more frequent enzyme-substrate complex formation
Substrates and enzymes also collide with more energy, making it more likely for bonds to be formed or broken (allowing the reaction to occur)
denaturation
If temperatures continue to increase past a certain point, the rate at which an enzyme catalyses a reaction drops sharply, as the enzyme begins to denature:
The increased kinetic energy and vibration of the enzyme molecules puts a strain on them, eventually causing the weaker hydrogen and ionic bonds that hold the enzyme molecule in its precise shape to start to break
The breaking of bonds causes the tertiary structure of the protein (i.e. the enzyme) to change
The active site is permanently damaged and its shape is no longer complementary to the substrate, preventing the substrate from binding
Denaturation has occurred if the substrate can no longer bind
the effect of temperature on the rate of an enzyme-catalysed reaction
The optimum temperature of an enzyme and the temperature at which an enzyme is denatured varies according to the habitat to which an organism is adapted
Most enzymes present in living organisms denature at temperatures above 60 °C
Very few human enzymes can function at temperatures above 50 °C
Humans maintain a body temperature of about 37 °C and even temperatures exceeding 40 °C can cause the denaturation of some enzymes
Some bacteria that live in thermal springs have enzymes that can withstand temperatures in excess of 80 °C
These enzymes are thermostable
temperature co efficient
The temperature coefficient for a biological reaction is the ratio between the rates of that reaction at two different temperatures
For most enzyme-catalysed reactions the rate of the reaction doubles for every 10 °C increase in temperature
The temperature coefficient (Q) for a reaction that follows this pattern is: Q₁₀ = 2
The temperature coefficient can be calculated using the following equation:
Temperature coefficient = (rate of reaction at (x + 10) °C) ÷ (rate of reaction at x °C)
enzyme concentration
Enzyme concentration affects the rate of reaction
The higher the enzyme concentration in a reaction mixture, the greater the number of active sites available and the greater the likelihood of enzyme-substrate complex formation
As long as there is sufficient substrate available, the initial rate of reaction increases linearly with enzyme concentration
If the amount of substrate is limited, at a certain point any further increase in enzyme concentration will not increase the reaction rate as the amount of substrate becomes a limiting factor
enzyme substrate concentration
The greater the substrate concentration, the higher the rate of reaction:
As the number of substrate molecules increases, the likelihood of enzyme-substrate complex formation increases
If the enzyme concentration remains fixed but the amount of substrate is increased past a certain point, however, all available active sites eventually become saturated and any further increase in substrate concentration will not increase the reaction rate
When the active sites of the enzymes are all full, any substrate molecules that are added have nowhere to bind in order to form an enzyme-substrate complex
For this reason, in the graph below there is a linear increase in reaction rate as substrate is added, which then plateaus when all active sites become occupied
reversible inhibitors
An enzyme’s activity can be reduced or stopped, temporarily, by a reversible inhibitor
There are two types of reversible inhibitors:
Competitive inhibitors have a similar shape to that of the substrate molecules and therefore compete with the substrate for the active site
Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape of the active site and therefore prevents the substrate from binding to it
reversible inhibitors and reaction rate
Both types of reversible inhibitors slow down or stop enzyme activity, decreasing the rate of reaction
Increasing the concentration of an inhibitor, therefore, reduces the rate of reaction and eventually, if inhibitor concentration continues to be increased, the reaction will stop completely
For competitive inhibitors, countering the increase in inhibitor concentration by increasing the substrate concentration can increase the rate of reaction once more (more substrate molecules mean they are more likely to collide with enzymes and form enzyme-substrate complexes)
For non-competitive inhibitors, increasing the substrate concentration cannot increase the rate of reaction once more, as the shape of the active site of the enzyme remains changed and enzyme-substrate complexes are still unable to form
end product inhibition and the control of metabolic pathways
Reversible inhibitors can act as regulators in metabolic pathways
Metabolic reactions must be very tightly controlled and balanced, so that no single enzyme can ‘run wild’ and continuously and uncontrollably generate more and more of a particular product
Metabolic reactions can be controlled by using the end-product of a particular sequence of metabolic reactions as a non-competitive, reversible inhibitor:
As the enzyme converts the substrate into product, the process is itself slowed down as the end-product of the reaction chain binds to an alternative site on the original enzyme, changing the shape of the active site and preventing the formation of further enzyme-substrate complexes
The end-product can then detach from the enzyme and be used elsewhere, allowing the active site to reform and the enzyme to return to an active state
This means that as product levels fall, the enzyme begins catalysing the reaction once again, in a continuous feedback loop
This process is known as end-product inhibition
non reversible inhibitors
Some inhibitors can form covalent bonds with enzymes, inhibiting them permanently
These are known as non-reversible or irreversible inhibitors
If this type of inhibition occurs in a living cell or organism it will result in the complete inactivation of the enzyme
This can be dangerous as can cause the biological reaction the enzyme is catalysing to be completely stopped
The only way to avoid this is for the cell or organism to produce more of the enzyme being inhibited, which can only be achieved by transcribing and translating the gene(s) for that enzyme, which is a relatively slow process
This is why some non-reversible inhibitors are considered to be metabolic poisons
For example, cyanide acts as a non-reversible inhibitor of cytochrome oxidase, a mitochondrial enzyme that catalyses one of the key reactions in aerobic respiration
This can be fatal as it takes too long to produce new enzymes and the organism will die before this can occur
As it stops a metabolic reaction, cyanide is known as a metabolic poison
Other non-reversible inhibitors, such as lead and mercury, are also serious poisons
For example, lead acts as a non-reversible inhibitor of ferrochelatase, an enzyme involved in the production of haem for haemoglobin
Some non-reversible inhibitors can be beneficial if they can be used, in a medical context, to inhibit enzymes that cause harm to some individuals
cofactors
There are substances other than substrates and inhibitors that interact with enzymes
Some enzymes can only function properly if another non-protein substance is present
For example, some enzymes are inactive until they combine with a non-protein substance that changes their tertiary structure (allowing the active site to bind correctly with the substrate)
These substances are broadly known as cofactors
Some enzymes require inorganic ions to function properly
Particular inorganic ions may help to stabilise the structure of the enzyme or may actually take part in the reaction at the active site
For example, chloride ions act as a cofactor for amylase
This means that in order for amylase to be able to digest starch into maltose, chloride ions must be present
The inorganic ions that an enzyme requires in order to function are known as inorganic cofactors
coenzymes
Larger organic (carbon-containing) cofactors are known as coenzymes
Some coenzymes are permanently bound to the enzyme they assist, often in or near the active site
Some coenzymes only bind temporarily during the reaction
Coenzymes link different enzyme-catalysed reactions into a sequence during metabolic processes, such as photosynthesis and respiration
Vitamins are an important source of coenzymes. For example, many vitamins in the B vitamin group are used in the production of important coenzymes, including:
Pantothenic acid, a key component of coenzyme A (a coenzyme required for the oxidation of pyruvate during the link reaction that occurs between the glycolysis and Krebs cycle stages of respiration)
Nicotinic acid, used to produce the coenzymes NAD and NADP (coenzymes required in many different metabolic reactions, including many of the reactions that take place during photosynthesis and respiration)
Vitamin B₁ (riboflavin), used to produce the coenzyme FAD (a coenzyme required in the Krebs cycle during respiration)
examples of co enzyme functions
During many of the reactions in respiration, the coenzymes NAD and FAD are alternately reduced and oxidised, transferring energy in the form of hydrogen ions
The coenzyme NADP fulfils this same role in chloroplasts during photosynthesis
The coenzymes ATP and coenzyme A act in a different way, by transferring chemical groups. For example:
ATP is responsible for the transfer of phosphate groups between respiration and energy-consuming processes in cells
Coenzyme A is responsible for the transfer of an acetyl group (-CH₃CO) from fatty acids and glucose during respiration
