chapter 2: enzymes!

Question 1

what are the general properties of enzymes?

Answer 1

1. enzymes are highly specific
   - an enzyme with absolute specificity only catalyses a single specific reaction in a single specific substrate
   - an enzyme with group specificity catalyses a single specific reaction in a group of related substrates
2. enzymes are highly efficient in very small amounts
   - enzymes have a high turnover number ( number of substrate molecules converted into products per unit time by one molecule of enzyme)
   - this is because the enzyme remains unchanged at the end of th reaction, and the active site can be used repeatedly
3. enzymes catalyse reversible reactions
   - enzymes speed up rate of both forward and backward reactions until equilibrium is reached in a reversible reaction
   > enzymes speed up the rate at which equilibrium is reached
   - enzymes do not alter position of equilibrium
   - the presence of enzymes also does not alter the properties of the end-products of the reaction they catalyse
4. enzymes are affected by reaction conditions
   - can be affected by various factors like enzyme concentration, substrate concentration, temperature, pH and inhibitors

Question 2

mode of enzyme action

what is the energy profile of a reaction?

Answer 2

• a chemical reaction involves both bond breaking and bond forming
• it requires energy to contort reactant molecules into a highly unstable state before bonds can be broken and new ones can be formed
• the initial investment of energy to contort reactant molecules so the bonds can break is known as free energy of activation, or activation energy (Ea)
• Ea is the energy barrier that has to be overcome before a reaction can occur and it determines the rate of reaction
  > higher activation energy will lead to a slower rate of reaction
• Ea is often supplied in the form of thermal energy (heat) absorbed by the reactant molecules from the surroundings

absorption of thermal energy can lead to:
- increase in speed of reactant molecules and thus an increase in frequency of collisions between reactant molecules
- thermal agitation of atoms within molecules, causing bonds to break more easily

• when the reactant molecules absorb sufficient energy for bonds to break, the reactants are known to reach an unstable condition known as the transition state
• once bonds are broken, atoms will settle into their new and more stable bonding arrangements and energy is released into the surrounding
• an enzyme works by lowering the activation energy of the reaction it catalyses
• increasing the rate of reaction and the rate at which equilibrium is reached

Question 3

mode of enzyme action

describe the enzyme structure and active site

Answer 3

1. an enzyme consists of four categories of amino acids
   - catalytic residues: make or break chemical bonds; responsible for the enzyme’s catalytic activity
   - contact/ binding residues: hold substrate(s) in place during catalysis, by forming temporary interactions like weak hydrogen bonds with the substrate
   -** structural residues:** maintain the correct specific 3D conformation of the active site so that enzyme can function properly
   - non-essential residues: near the surface of enzyme; no specific functions thus can be removed or replaced without loss of function
2. the active site of an enzyme is a small pocket/ groove/ cleft on its surface and is usually formed by only a few (3 to 12) amino acid residues
   - it has a specific 3D conformation which is complementary to that of its substrate
   - the active site contains catalytic and binding residues and the site of catalysis

Question 4

what are the three steps in the enzyme’s mode of action?

Answer 4

step 1: formation of enzyme-substrate complex
- both the specific 3D conformation and charges present at the active site allow only specific substrates with a 3D conformation and charges that are complementary to the active site to enter it in a specific orientation
- a small rearrangement (induced fit) of chemical groups may occur in both the enzyme active site and the substrate molecules
- certain binding residues of the active site may form weak, temporary but extensive bonds with substrates, forming the enzyme-substrate complex
- weak bonds are usually hydorgen bonds

step 2: catalysis / lowering of activation energy
- interactions between the enzyme ans substate molecules occur, lowering tthe activation enerfy and increasing the rate of the enzyme-catalysed reaction

lowering the activation may occur by:
- bringing substrates close together with each other at the active site
- bringing substrates into the correct orientation with each other to react
- altering the charges on the substrates
- physically straining bonds within substrates
- creating a more conducicen micro-environment for substates to react

step 3: product formation and regeneration of enzymes
- when catalysis is completed, the product(s) are no longer complementary in 3D conformation and charge to the active site and thus leave the active site
- the temporary changes in 3D conformation and charge at the active site are reversed and the active site is now availible again for substrate binding

Question 5

enzyme specificity

how does the ‘lock and key’ hypothesis explain why enzymes are specific?

Answer 5

• the enzyme’s (lock) active site has a fixed 3D conformation produced by the folding of the polypeptide chain
• this fixed 3D conformation is perfectly complementary in shape to the substrate (key)

Question 6

enzymes specificity

how does the induced fit hypothesis explain enzyme speecificity?
- this hypothesis states that the active site is ____ ____
- the active site is able to ____ itself around the substrate
- an enzyme thus not a still structure that is of fixed 3D conformation
- > the active site is also not a ____ ____ to hold the substrate
- the 3D conformation of the active site of the enzyme molecule is initially not complementary to the 3D conformation of substrate
- the substrate entering the active site induces a slight change in the active site’s 3D conformation due to interactions between the substrate’s ____ ____ and the R groups of the amino acid residues that form the ____ ____
- the 3D conformation change causes the active site to fit more ____ around the substrate
- the shape changes in the active site to fit the substrate molecule better is called ____ ____
- ____ fit brings catalytic residues into ____ that improve their ability to catalyse the chemical reaction
- after products are formed, they no longer fit into the active site and leaves the active site
> ( no longer complementary in 3D conformation and charge)
> the enzyme molecule ____ to its original conformation and it is free to bind another substrate molecule

Answer 6

• this hypothesis states that the active site is physically flexible
• the active site is able to mould itself around the substrate
• an enzyme thus not a still structure that is of fixed 3D conformation
• > the active site is also not a rigid groove to hold the substrate
• the 3D conformation of the active site of the enzyme molecule is initially not complementary to the 3D conformation of substrate
• the substrate entering the active site induces a slight change in the active site’s 3D conformation due to interactions between the substrate’s chemical groups and the R groups of the amino acid residues that form the active site
• the 3D conformation change causes the active site to fit more snugly around the substrate
• the shape changes in the active site to fit the substrate molecule better is called** induced fit**
• induced fit brings catalytic residues into positions that improve their ability to catalyse the chemical reaction
• after products are formed, they no longer fit into the active site and leaves the active site
  > ( no longer complementary in 3D conformation and charge)
  > the enzyme molecule revert to its original conformation and it is free to bind another substrate molecule

Question 7

rate of enzyme-catalysed reactions

how can we measure the rate of enzyme-catalysed reactions?

Answer 7

• can be measure by the amount of substrate depleted or used over time
• or the amount of products formed over time
• the actual rate of reaction can be calculated by measuring the slope of the tangent at the initial part of the curve
• the steeper the slope, the higher the rate

Question 8

rate of enzyme-catalysed reactions

what are some factors affecting the rate of enzyme-catalysed reactions?

Answer 8

-enzyme concentration, substrate concentration, temperature and pH
> when considering one factor, other factors must be kept constant

Question 9

factors affecting the rate of enzyme-catalysed reactions

how does enzyme concentration affect the rate of enzyme-catalysed reactions?

( what happens when enzyme concentration increases)

Answer 9

• when the enzyme concentration is increased, the rate of reaction increases proportionally to the increase in enzyme concentration
• this is because there are more active sites available for substrates to bind at
  > leading to an increase in the frequency of effective collisions between enzymes and substrates
  > increase in the rate of enzyme-substrate complex formation and
  > the increase in the rate of product formation
• provided that temperature and other physical conditions are kept constant during the reaction and the substrates are in excess
  > the rate of reaction is directly proportional to enzyme concentration
• if the substrate concentration is limiting, further addition of enzyme will not lead to an increase in the reaction rate
  > there are many more empty active sites that there are substrates availible

Question 10

factors affecting the rate of enzyme-catalysed reactions

**how does substrate concentration affect the rate of enzyme-catalysed reactions?
**
- the reaction for most enzyme-controlled reactions varies with the availibility of substrate

• when the enzyme concentration is in excess, an increase in the substrate concentration will lead to an ____ in the rate of reaction
• at low substate concentrations, the rate of the reaction ____ with increasing substrate concentrations
  > many enzyme molecules have availible active sites which are ____ and the limited number of substrate molecules largely determines the reaction rate
  > substrate concentration is the limiting factor
• increasing substrate concentration will increase the reaction rate as more active sites are being occupied and more ____ ____ ____ and products are formed per unit time
• beyond a certain high substrate concentration, when there are too many substrate molecules, the rate of reaction will remain ____ as all the active sites are being occupied by substrate molecules all at once
• all the active sites are saturated
• this will be the theoretical maximum rate of reaction for that enzyme and is reffered to as its Vmax
• beyond this substrate concentration and the rate of reaction is still constant,
  > an increase in rate can only be achieved by increasing ____ concentration
  > substrate concentration is thus no longer the limiting factor, with ____ concentration now being the limiting factor

Answer 10

• the reaction for most enzyme-controlled reactions varies with the availibility of substrate
• when the enzyme concentration is in excess, an increase in the substrate concentration will lead to an increase in the rate of reaction
• at low substate concentrations, the rate of the reaction increases with increasing substrate concentrations
  > many enzyme molecules have availible active sites which are unoccupied and the limited number of substrate molecules largely determines the reaction rate
  > substrate concentration is the limiting factor
• increasing substrate concentration will increase the reaction rate as more active sites are being occupied and more enzyme substrate complexes and products are formed per unit time
• beyond a certain high substrate concentration, when there are too many substrate molecules, the rate of reaction will remain constant as all the active sites are being occupied by substrate molecules all at once
• all the active sites are saturated
• this will be the theoretical maximum rate of reaction for that enzyme and is reffered to as its Vmax
• beyond this substrate concentration and the rate of reaction is still constant,
  > an increase in rate can only be achieved by increasing enzyme concentration
  > substrate concentration is thus no longer the limiting factor, with enzyme concentration now being the limiting factor

Question 11

how does temperature affect the rate of reaction?
(below optimum temperature)
(and what happens when there in an increase in temperature?)

Answer 11

• at very low temperatures, the enzymes are inactive
• rate of reaction is very low as the frequency of effective collisions between substrate and enzyme molecules is low
• as there is an increase in temperature, both the enzyme and substrate molecules gain kinetic energy
• there is an increase in frequency of effective collisions between substrate and enzyme molecules
• more ES complexes are formed per unit time and therefore more products are formed per unit time
• rate of reaction increases until optimum temperature is reached

Question 12

how does temperature affect the rate of reaction?
(at optimum temperature)

Answer 12

• rate of reaction is fastest as there is greatest freqeuncy of effective collsions between enzymes and substate molecules
• rate of ES complex formation is the highest

Question 13

how does temperature affect the rate of reaction?
(above optimum temperature)

Answer 13

• rate of reaction decreases despite an increase in the frequency of collisions
• increasing temperature increases thermal agitation of enzyme molecules
• this casues R group interactions like hydrogen bonds, ionic bonds and hydrophobic interactions in tertiary and quaternary structures to be disrupted
• hydrogen bonds in the secondary structures may also be disrupted
• enzyme molecules are denatured as the precise 3D conformation of the active site is changed
  > so no longer complementary in 3D conformation to the substrate and cannot bind the substrate
• catalytic activity of the enzyme is lost, leading to a lower rate of ES complez formation and thus lower rate of reaction ( product formation)
• disulfide bonds are strong covalent bonds are usually broken only at very high temperatures by high heat energy but protein denaturation usually occurs before it can happen

Question 16

at optimum pH for enzymes:
- the rate of reaction is ____ as the 3D conformation of active site is most ideal for ____ binding and frequency of ____ ____ between enzymes and substrates is the highest
- ____ bonds (like ionic bonds, hydrogen bonds between R groups) stabilise the ____ structure of the enzyme
- ____ residues and ____ residues in the active site are of the correct charge to bind to the substrate molecules and catalyse the reaction respectively

Answer 16

• the rate of reaction is highest as the 3D conformation of active site is most ideal for substrate binding and frequency of effective collisions between enzymes and substrates is the highest
• intramolecular bonds (like ionic bonds, hydrogen bonds between R groups) stabilise the tertiary structure of the enzyme
• contact residues and catalytic residues in the active site are of the correct charge to bind to the substrate molecules and catalyse the reaction respectively

Question 17

unlike the effects of heat on enzymes, the effects of pH on enzyme are normally ____
- when pH changes are within limits
- restoring the pH to the optimum level usually ____ the normal enzyme activity and thus the normal rate of reaction

• however, if pH deviates drastically from the ____ pH, the enzymes are ____ denatured
• adjusting the pH will not restore the rate of reaction

Answer 17

unlike the effects of heat on enzymes, the effects of pH on enzyme are normally reversible
- when pH changes are within limits
- restoring the pH to the optimum level usually restores the normal enzyme activity and thus the normal rate of reaction
​
- however, if pH deviates drastically from the optimum pH, the enzymes are permanently denatured
- adjusting the pH will not restore the rate of reaction

Question 18

what are the two types of enzyme inhibition?

Answer 18

1. competitive inhibition
2. non-competitive inhibition

Question 19

describe the proccess of competitive inhibition.
(shape of the inhibitor)
(what type of bonds formed with the enzyme)
( how the reaction is affected)

Answer 19

1. the inhibitor molecule has a similar 3D conformation as the substrate molecule and is thus also complementary in 3D conformation to the enzyme’s active site and is able to bind to it
2. the competitive inhibitor therefore competes with the substrate for limited number of active sites
3. the competitive inhibitor binds reversibly to the active sites
   - only** weak bonds** (like hydrogen bonds form between the inhibitor and the enzyme)
   - the effect is temporary and causes no permanent damage to the enzyme
   - the 3D conformation of the active site is not altered
   - removal of the inhibitor restores enzyme activity
4. when the competitive inhibitor enters the active site, it prevents substrates from binding at the active site
   - fewer ES complexes and thus products are formed per unit time, reducing the reaction rate
   - this is especially so at low substrate concentrations

Question 20

can competitive inhibition be overcomed? if yes, how?

Answer 20

• competitive inhibition can be overcome by increasing substrate concentration
• at higher substrate concentrations, there is a higher probability a successful collision between a substrate and enzyme molecule rather than an inhibitor and an enzyme molecule
• the substrate molecules can thus compete more effectively with the inhibitor molecules for the limited number of enzymes active sites

Question 21

describe the process of non-competitive inhibition
( shape of inhibitor)
(where it binds)
(reversible?)

Answer 21

• in non-competitive inhibition, the inhibitor is not structurally similar to the substrate molecule, and therefore does not bind at the active site
• it will bind to the enzyme at an allosteric site which is located away from the active site
• by binding at the allosteric site, the non-competitive inhibitor causes a** change in enzyme structure**
• specifically the 3D conformation of the active site is altered such that it is no longer complementary and able to bind to the substrate
• reaction rate is thus decreased

non-competitive inhibitors can either bind reversibly or irreversibly at the allosteric site
- in reversible binding, the non-competitive inhibitor forms **weak and temporary bonds **
- (like hydrogen bonds)
- a certain portion of the total number of enzymes are temporarily inhibited

• in irreversible binding, the non-competitive inhibitor forms strong and permanent covalent bonds with the allosteric site
• a certian proportion of the total number of enzymes is permanently inhibited at the start of the reaction

Question 22

can nono-competitive inhibition be overcome?

Answer 22

• unlike competitive inhibition, an increase in the substrate concentration will not eventually overcome the effect of non-competitive inhibition
• by binding at the allosteric site, the inhibitor effectively lowers the number of active sites availible for substrate binding constantly and onxe it is introduced
• a non-competitive inhibitor therefore lowers the effective enzyme concentration
• ## even at high substrate concentrations, V max of the uninhibited reaction is still not achieved for the case of non-competitive inhibition

Question 23

what are allosteric enzymes?

Answer 23

• the activities of allosteric enzymes can be regulated by the attachment of a regulatory molecule at their allosteric site which is away from the active site
• allosteric enzymes typically have multiple active sites located on different protein subunits

Question 24

what happens when an allosteric inhibitor binds to an allosteric enzyme?

Answer 24

• when an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits change their conformation slightly so that they are not complementary to that of the substrates’ conformation
• the inactive form is stabilised
• allosteric inhibition is a form of non-competition inhibition

Question 25

what happens when an allosteric activator binds to an allosteric enzyme?

Answer 25

- when an allosteric activator binds to an enzyme, all active sites on the protein subunits change their conformation slighly - so that they become complementary to that of the substrates' conformation - the active form is stabilised

Question 26

**allosteric enzymes exhibit cooperativity** - in cooperativity, a substrate molecule binding to one active site in a ____ allosteric enzyme triggers in a ____ ____ in all the other subunits - thereby increasing catalytic activity at the other active sites - one substrate molecule increases an ____ ____ to additional substrate molecules and ____ the other enzymatic sub-units to act on additional substrate molecules more readily - the bidning of the first substrate molecule to an active site changes the 3D conformation of the ____ ____ ____ in a multi-subunit enzyme - causing them to become more ____ in 3D conformation to substrate molecules and hence bind better to them

Answer 26

- in cooperativity, a substrate molecule binding to one active site in a **multi-subunit** allosteric enzyme triggers in a **shape change** in all the other subunits - thereby increasing catalytic activity at the other active sites ​ - one substrate molecule increases an **enzyme's affinity**to additional substrate molecules and **primes** the other enzymatic sub-units to act on additional substrate molecules more readily ​ - the binding of the first substrate molecule to an active site changes the 3D conformation of the **other active sites**in a multi-subunit enzyme - causing them to become more **complementary** in 3D conformation to substrate molecules and hence bind better to them

Question 27

**end product inhibition:** - end product inhibition / feedback inhibition, provides a flexible way to self regulate enzyme pathways - the final product acts as a ____ non-competitive/allosteric inhibitor of the enzyme at the ____ of a metabolic pathway - the enzyme catalysisnt the first step in a biochemical pathway often has an ____ ____ to which the end product produced at the last step of the metabolic pathway binds - when the final product of the pathway starts to ____, the metabolic pathway leading to its manufacture will be ____ shtut down as it is not needed since the final product is in excess - in end-product inhibition, tbe end product itself inhibits the ____ enzyme that leads to its production - this serves as a form of ____, preventing over production of the product and hence ____ of resources - as the end-production is used up, the inhibition is ligted and the production of the end product is switched back on

Answer 27

- end product inhibition / feedback inhibition, provides a flexible way to self regulate enzyme pathways - the final product acts as a **reversible** non-competitive/allosteric inhibitor of the enzyme at the **beginning** of a metabolic pathway - the enzyme catalysisnt the first step in a biochemical pathway often has an **allosteric site** to which the end product produced at the last step of the metabolic pathway binds - when the final product of the pathway starts to **accumulate**, the metabolic pathway leading to its manufacture will be **temporarily** shtut down as it is not needed since the final product is in excess - in end-product inhibition, tbe end product itself inhibits the **initial** enzyme that leads to its production - this serves as a form of **self-regulation**, preventing over production of the product and hence **wastage** of resources - as the end-production is used up, the inhibition is ligted and the production of the end product is switched back on