C4 — Enzymes Flashcards
Activation energy definition
The minimum amount of energy required to start a chemical reaction.
Holoenzyme definition
A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions.
Rate of an enzyme-catalysed reaction definition?
The amount of substrate which is converted to product by enzymes per unit time.
Allosteric regulation definition
The regulation of an enzyme by the binding of molecules at an allosteric site, ie. a site other than the active site.
Metabolism definition
The totality of an organism/cell’s chemical reactions.
Vmax (Maximal reaction velocity) definition
The maximum rate that a reaction can proceed in the presence of a specific concentration of enzyme and excess substrate.
Km definition
The substrate concentration that allows an enzyme-catalysed reaction to proceed at half the maximum velocity, ½ Vmax.
- Measured in substrate concentration.
- Represents the affinity of an enzyme for a particular substrate
- enzymes with a low Km value exhibit a high affinity for their substrate;
- enzymes with a high Km value exhibit a low affinity for their substrate.
Explain how the extra 44 amino acids prevent pepsin from digesting proteins in the cell. [2]
- Extra 44 amino acids occupies active site/covers active site of pepsin
- Blocks the active site such that proteins of the cell (substrate) are unable to bind hence no formation of enzyme-substrate complex
Suggest why protein-digesting enzymes are synthesised as inactive pro enzymes inside cells. [1]
- Active enzymes cause damage to the protein structure of cells that produced them
- Active enzymes cause damage to secreting cells
- Active enzymes cause damage to surrounding tissues
Explain why the enzyme is not active at pH8. [3]
- At pH 8, the ionic charge of the R groups of amino acid residues are altered, hence disrupting the ionic and hydrogen bonds
- Chymos is denatured at pH 8, loss of specific 3D conformation of the active site of enzyme
- Active site is no longer complementary to the substrate, preventing enzyme-substrate complex formation
Describe, with reference to figure 2.1, the effect of temperature on the rate of protein digestion by thermitase. [3]
- Ref. To temperature increase from 10dgC to 76dgC, rate of protein digestion gradually increase from 0au to 650au /max tempt from 75 to 77dgC/ max rate of protein digestion from 640au to 660au
- Ref. To optimum tempt being 76dgC, max rate of reaction at 650au
- ref. To tempt increase from 76dgC to 90dgC/beyond 76dgC, rate of protein digestion rapidly decrease from 650au to 350 au
- Reject descriptions from 0dgC to 10dgC
Explain the effect on thermitase of increasing the temperature above 80dgC. [3] (Covalent bonds not broken)
- Ref. To denaturation of thermitase
- Ref. To increased thermal agitation, more kinetic energy needed to overcome the R group interactions
- ref. To Disruption of any 2 of the following R-group interactions: hydrogen bonds/ionic bonds/hydrophobic interactions
- resulting in loss of specific 3D conformation of active site, such that the active site is not complementary to the substrate/ enzyme substrate complex cannot be formed
- Hence, rate of reaction will decrease as thermitase cannot catalyse protein digestion
Modified subtilisin is similar to subtilisin, but has had 8 of its amino acids replaced with different amino acids. Describe AND explain the effect of this modification on the activity of subtilisin. [4]
(Must compare data betw subtilisin and modified subtilisin)
Describe: (must hv 2 to get 1m)
- Ref. To higher maximum rate of protein digestion at 320 at for modified subtilisin, compared to 80 at for subtilisin
- ref. To higher optimum tempt of 76dgC for modified subtilisin, compared to 58dgC for subtlisin
Explain:
- Ref. To R groups of the 9 amino acids having different size/charge/polarity
- ref. To ability to form stronger bonds such as disulfide bonds/ref. To ability to form MORE non-covalent bonds eg hydrogen bonds/ionic bonds/hydrophobic interactions
- ref. To increased thermostability of the enzyme ie higher optimum temperature/increased affinity of enzyme for substrate ie higher maximum rate
Explain why in the reaction with enzyme only, as substrate concentration increases:
(I) The rate of reaction increases at first: [2]
- At low substrate concentrations, substrate concentration is limiting hence not all enzyme active sites are occupied/enzymes with unoccupied active sites are available [1]
- An increase in substrate concentration will allow these enzymes with unoccupied sites to bind to substrate hence increasing the frequency of effective collisions between the enzyme active sites and substrate molecules [1]
- An increase in substrate concentration increases the rate of enzyme-substrate complexes formation [1]
Explain why in the reaction with enzyme only, as substrate concentration increases:
(II) The rate of reaction becomes constant: [2]
- At high substrate concentrations, substrate concentration is no longer limiting and enzyme concentration is now limiting. [0.5] +
- At any one time, as all enzyme active sites are occupied, any added substrate molecules will be unable to bind to any active site [0.5]
- The frequency of effective collisions between the enzyme active sites and substrate molecules is at its maximum [1] OR
- The rate of enzyme-substrate complexes formation is at its maximum
Explain why in figure 2.1, the addition of a non-competitive inhibitor causes the reaction to become constant at a lower rate.
- The non-competitive inhibitor binds to a site other than the active site of the enzyme (REJECT allosteric site)
- which causes a change in specific 3D conformation of the enzyme’s active site thus preventing substrate molecules from binding
- a certain proportion of the enzyme molecules are rendered inactive resulting in a lower Vmax
Suggest why the penicillin molecule is an effective inhibitor of transpeptidase. [3]
- The penicillin molecule is structurally similar to the cell wall peptide
- and competes with the cell wall peptide for binding to the transpeptidase active site
- The penicillin molecule binds to the transpeptidase active site via covalent bonds thus irreversibly inhibiting transpeptidase.
Effects and properties of enzymes
Effect of enzymes:
the activation energy is lowered in an enzyme-catalysed reaction
more reactant molecules can surmount the energy barrier to reach the transition state to be converted into product molecules
the total energy difference / free energy change or Gibbs free energy (triangle G) between the reactant molecules and product molecules remains the same.
Properties of enzymes:
1. Effective in small amounts, remain chemically unaltered at the end of the reaction and can be reused.
2. Enzymes are extremely efficient. Enzyme-catalysed reactions proceed 103 to 108 times faster than uncatalysed reactions.
3. Enzymes have a high degree of specificity. Most enzymes are specific to one type of substrate molecule. Other enzymes are specific to a group of similar substrates.
4. Enzymes can be denatured by heat and they act most efficiently at an optimum temperature.
5. Enzymes are affected by pH and they act most efficiently at their optimum pH.
6. Enzymes activity can be regulated by activators and inhibitors.
4 types of amino acid residues and their functions
Catalytic amino acid residues
The R groups of these amino acids are directly involved in the
catalytic activity, ie. making or breaking of chemical bonds once the substrate is bound.
Binding amino acid residues
The R groups of these amino acids hold the substrate(s) in position via non-covalent bonds while catalysis takes place.
Structural amino acid residues
Involved in maintaining the specific 3D conformation of the active site, as well as the enzyme as a whole.
Non-essential amino acid residues
Have no specific functions, can be removed or replaced without the loss of the enzyme’s catalytic function
Types of cofactors
Note: apoenzyme + cofactors = holoenzyme
Inorganic metal ions
- Mostly small divalent ions eg. Ca2+
- May either be a component of the active site or affect enzyme activity through allosteric regulation. Allosteric enzymes have multiple subunits and through conformational changes, bind activators of inhibitors at sites other than the active site.
- They usually bind reversibly to the enzyme and act by altering the enzyme’s active and/or allosteric sites to facilitate the catalytic reaction carried out by the enzyme.
Coenzymes
- Loosely associates with the enzyme during the reaction.
- Coenzymes act as transient carriers of specific functional groups, hydrogen or electrons. Most coenzymes are derived from vitamins.
Prosthetic group
- Tightly bound to the enzyme on a permanent basis.
Formation of enzyme substrate complex:
- When enzyme and substrate collide in the correct orientation, an effective collision occurs, the substrate will be bound to the enzyme at the enzyme’s active site.
- An enzyme-substrate complex is formed.
- The substrate molecule is held in the active site by non-covalent bonds such as hydrogen and ionic bonds between the R groups of the binding amino acids and the substrate molecule
- The R groups of the catalytic amino acid residues at the active site catalyse the conversion of the substrate to product.
- The alteration in chemical conformation results in the product molecule being released from the active site as it is no longer complementary to the active site structure.
- The enzyme active site is free for the binding of another substrate molecule (ie. the enzyme can be reused).
How enzyme lowers activation energy:
- orientating the substrates in close proximity, in the correct orientation, to undergo chemical reactions.
- straining critical bonds in the substrate molecule(s), allowing the substrates to attain their unstable transition state.
- providing a microenvironment that favours the reaction (eg. the presence of specific amino acids / ions at the active site may result in a specific set of molecular conditions that favours the formation / breakage of particular bonds).
Lock and key hypothesis vs induced fit hypothesis
Lock and key hypothesis:
The enzyme is viewed as a rigid structure, where only substrates that are exactly
complementary to the conformation of the active site are able to bind to the active site for catalysis.
Induced fit hypothesis:
Active site flexibility, i.e. active site
- does not have a rigid conformation that fits only one type of substrate
- is rather flexible in conformation and can allow more than one type of substrate to bind is not in precise complementary conformation to the substrate before binding to the substrate
Upon binding of substrate, the active site changes its conformation slightly to bind the substrate even more firmly/snugly so that the R groups of the catalytic amino acids at the active site are:
- moulded into a specific conformation
- brought into close proximity to the chemical bonds in the substrate hence facilitating catalysis where the substrate is converted to product
How to measure rate of enzyme-catalysed reaction
Product formation:
Measuring the rate of oxygen production (easier) or the rate of water production in a breakdown reaction from H2O2 (hydrogen peroxide) to water and oxygen by the enzyme catalase
2 H2O2 (aq) —(catalase)-> 2 H2O (l) + O2 (g)
Substrate usage:
Measuring the rate of disappearance of starch (easier) or the rate of maltose production in a breakdown reaction of starch to maltose by enzyme amylase
Starch —(amylase)-> maltose.
The principle of limiting factors states that:
- The rate of a biochemical process, which consists of a series of reactions, is limited by the slowest reaction in the series.
- When a biochemical process is affected by several factors, its rate is limited by that factor which is in the shortest supply, known as the limiting factor.
- When the supply of the limiting factor is increased, it will lead to an increase in the rate of reaction.
Factors affecting rate of enzyme reaction: substrate concentration
Low:
Increase in substrate concentration results in a proportional increase in the rate of reaction
(substrate concentration is the limiting factor).
- Not all the active sites of the enzymes are occupied.
- Rate is limited by the concentration of substrate.
- An increase in substrate concentration increases the frequency of effective collisions between the enzyme active site and substrate molecules, hence increasing the number of enzyme-substrate complexes formed per unit time and consequently the amount of product formed per unit time, resulting in a proportional increase in the rate of reaction.
High:
A point will be reached when any further increase in substrate concentration does not result in an increase in the rate of reaction (i.e. the graph reaches a plateau).
- The active site of every enzyme molecule is occupied at any given moment. Rate is limited by saturation of enzyme active sites.
- Enzyme concentration is limiting.
- Any added substrates have to ‘wait’ until existing E-S complexes dissociate to release their products and free enzyme molecules in order to form new E-S complexes.
- Further increase in substrate concentration does not result in an increase in the rate of reaction ie. graph reaches a plateau.
- Substrate concentration is no longer the limiting factor.
- Rate of reaction can only increase with the addition of enzymes.
(RMB graph)
Factors affecting rate of enzyme reaction: enzyme concentration
The turnover number (Kcat) is the maximum number of molecules of substrate that an
enzyme can convert to product per catalytic site per unit time.
Low:
Increase in enzyme concentration results in a proportional increase in the rate of reaction ie. concentration of enzyme is the limiting factor.
- The increase in enzyme concentration provides more active sites, and therefore
- Increasing the frequency of effective collisions between substrates and active sites.
- More enzyme-substrate complexes formed per unit time
- Resulting in an increase in the amount of product formed per unit time
- Hence, resulting in an increase in rate of reaction.
High:
A point will be reached when any further increase in enzyme concentration does not
result in an increase in the rate of reaction ie. the graph reaches a plateau
- Enzyme concentration is no longer a limiting factor. There are not enough substrate molecules competing for the active sites available.
- Substrate concentration is limiting.
- Rate of reaction can be increased with the addition of substrate
Factors affecting rate of enzyme reaction: temperature
The dual effect of temperature on enzyme activity refers to the effect of heat,
- increasing effective collision of the reactants and
- decreasing the stability of the enzyme protein
Resulting combination determines the optimum temperature of enzyme.
As the temperature increases to the optimum temperature from 5°C to 40°C:
- At low temperatures near or below freezing point, enzymes are inactivated
- Increasing temperature increase the kinetic energy of the substrate and enzyme molecules, thereby
- increasing the frequency of effective collisions between substrates and active sites, increasing the formation of enzyme-substrate complexes per unit time and the amount of product formed per unit time.
- This increases the rate of reaction.
- Enzyme activity is highest at its optimum temperature of 40°C for most cases.
- The rate of ES complexes formation is the highest at optimum temperature
If the temperature is increased beyond the optimum temperature of 40°C:
- A decrease in the rate of reaction occurs despite the increasing frequency of collisions.
- Thermal agitation of enzyme molecule disrupts the hydrogen bonds, ionic bonds and other non-covalent interactions that stabilise the specific 3D conformation of the protein molecule.
- Loss in the 3D conformation of the enzyme and that of its active site so that there is no longer a complementary fit with the substrate.
- The enzyme is said to be denatured and loses its catalytic function.
- the frequency of effective collisions between substrates and active sites decrease and the rate of formation of enzyme-substrate complexes drops
- less product is formed per unit time.
Temperature coefficient, Q10
= rate of reaction at (x+10)dgC/ Rate of reaction at x dgC.
Q10 is the effect of temperature on chemical reactions.
In most reactions, rate of reaction doubles for every 10dgC increase in temperature between 10dgC and 40dgC ie. below optimum temperature, Q10 = 2.
Factors affecting rate of enzyme reaction: pH
Measure of the concentration of H+ (hydrogen ions) in a solution
Low pH: high concentration of H+ ions
High pH: low concentration of H+ ions
pH changes can alter the ionic charge of the acidic and basic R groups:
- Lower pH»_space; more H+ ions available to neutralise negative charges present in the enzyme.
- Higher pH»_space; less H+ ions available to neutralise negative charges present in the enzyme.
Change in ionisation of amino acids disrupts the ionic bonds/ hydrogen bonds that maintain the 3D conformation of the enzyme, denaturing the enzyme:
Structural amino acid residues that help in the stabilisation of the shape of the enzyme:
- Conformation of the active site is no longer complementary to the substrate.
- No enzyme-substrate complex can be formed.
Binding amino acid residues at active sites:
- substrate cannot be held in its correct orientation in the active site for catalysis to occur
Catalytic amino acid residues at active sites:
- R groups on catalytic amino acid residues no longer possess the correct ionisation/ charge to catalyse the required reaction.
Optimum pH:
- Rate of reaction is maximum – intra-molecular bonds are intact and conformation of active site is ideal for binding, therefore frequency of effective collision is the highest with largest amount of ES complexes formed.
- At pH values only slightly above and below the optimum pH, many enzymes will have a marked decline in enzyme activity as the 3D conformation is altered and affinity for the substrate is correspondingly decreased.
- Usually the pH of the environment in which the enzyme normally functions.
Activators
Activator binds to allosteric site of an enzyme, stabilising the active form of the enzyme and increases the affinity of the enzyme for substrate -> inducing a favourable conformation change in the active sites of all the subunits of the enzyme -> significantly amplifies the response of the enzyme to substrates -> amplification results in the sudden steep rise in the rate of reaction -> process is called allosteric activation
Inhibitors
Inhibitor binds to the same region, stabilising inactive form of the enzyme, decreasing affinity of the enzyme for its substrate.
How tightly an inhibitor binds to the enzyme affects the inhibition of an enzyme:
Reversible inhibition:
The inhibitor binds to the enzyme via weak non-covalent bonds such as hydrogen bonds, hydrophobic interactions.
The effect of the inhibitor is temporary, the inhibitor can be easily removed and cause no permanent damage to the enzyme.
When the inhibitor leaves, activity of the enzyme will be restored to normal.
Irreversible inhibition:
The inhibitor binds to the enzyme via covalent bonds.
The inhibition causes permanent damage to the enzyme molecule so that it is unable to carry out catalytic activity.
Enzyme inhibition
Competitive Inhibition: CI
Non-competitive inhibition: NCI
Structure of inhibitor:
CI: Structurally similar to the substrate molecule
NCI: No structural resemblance to substrate
Site of binding:
CI: Inhibitor competes with substrates to bind to active site, preventing substrate from binding to the active site
NCI: Inhibitor binds to enzymes at a region other than the active site, thus does not compete with the substrate for the active site.
Effect of increasing substrate concentration:
CI: Inhibition can be reversed because substrate molecules can out-compete the inhibitor for the active site
NCI: No effect
Maximum rate of reaction:
CI: The same Vmax is reached at a higher substrate concentration than without inhibitors. Both reactions reach the same Vmax When S is very high.
NCI: Vmax in the presence of inhibitor is less than that of reaction without, even at high substrate concentration
Change in Km value:
CI: Larger Km
NCI: Km is unchanged as the affinity of
the substrate for the normal
enzyme remains unaffected
Final amount of product formed:
CI: same
NCI: same
Description:
CI: The initial rate of reaction is reduced in the presence of a competitive inhibitor as a longer period of time is needed to produce the same amount of product.
NCI: The initial rate of reaction is reduced in the presence of non-competitive inhibitors.
Explanation
CI: An increase in substrate concentration reduces the effect of inhibition.
The substrate and the inhibitor are in direct competition for the enzymes’
active sites and the greater the proportion of the substrate molecules, the greater the chance a substrate can out-compete the inhibitor to enter the active site. -> a rate of reaction almost equivalent to Vmax can be attained.
-> The final amount of product formed is the same as the substrate continues to be converted by any enzyme molecules that are unaffected by the inhibitor.
NCI: The binding of a non-competitive inhibitor to a site other than the enzyme’s active site causes a change in 3D conformation of the enzyme’s active site -> prevents substrate molecules from binding. A certain proportion of the enzyme molecules are rendered inactive, Vmax is lower.
As the substrate and the inhibitor are not in direct competition for the same site, an increase in substrate concentration has no effect on the inhibition. The final amount of product formed is the same as the substrate continues to be converted by any enzymes molecules that are unaffected by the inhibitor
Advantages of metabolic pathways catalysed by enzymes:
- Biochemical reactions may proceed with no accumulation of products in the cells. Products become substrates of subsequent reactions.
- Reactants may be modified in a series of small steps, enabling:
A. energy to be released in controlled amounts or
B. minor adjustments to be made to the structure of molecules or
C. coupling of exergonic reactions to endergonic reactions - Each step is catalysed by a specific enzyme allowing for regulation and control of metabolism as each enzyme is a point for control of the overall pathway. Enzymes specific to the reaction are regulated by different inhibitors/activators, -> allowing for a finely balanced portioning of cell metabolites among different pathways.
- The steps in the pathway may be spatially arranged so that the product of one reaction is ideally located to become the substrate of the next enzyme. This permits the build-up of high local concentrations of substrate molecules and biochemical reactions can proceed rapidly. A pathway arranged in this way may be catalysed by a multi-enzyme complex.
- A multi-enzyme complex is made up of a team of enzymes for several steps of a metabolic pathway. The arrangement orders the sequence of reactions, as the product from the first enzyme becomes substrate of the adjacent enzyme in the complex, and so on until the end-product is released. This arrangement greatly increases the efficiency of the enzyme pathway.
End-product inhibition:
When the end-product of a metabolic pathway accumulates, it may act as an inhibitor and binds to the enzyme(s) of the preceding pathway, thus:
- alter the conformation of the active site of the enzyme and lower its affinity for its substrate
- block the entry of the substrate into the active site.
- Preventing or reducing further production of end-products
