ENZYMES 📈

Question 1

Activation energy

Answer 1

Minimum amount of energy required to start a reaction

Question 2

Catalyst

Answer 2

substance that increases the rate of a reaction while remaining chemically unchanged at the end of the reaction

Question 3

Enzyme

Answer 3

biological catalyst that catalyses all biochemical reactions by lowering the activation energy

activation energy lowered in enzyme-catalysed reaction, more reactant molecules are able to surmount energy barrier to reach transition state to be coverted to product moleucles

total energy difference or gibbs free energy change between reactants and products remain the same

Question 4

Analysis of energy profile diagram

Answer 4

Uphill portion: investment of energy required to start the reaction, activation energy

Peak: reactant molecules are in unstable transition state and bonds can be broken/formed

Downhill: after the appropriate bonds have been formed/broken, molecules settle into new bonding arrangement to form the product

Question 5

Properties of enzymes

6 points

Answer 5

1. effective in small amounts, can be resued as they are chemically unchanged at the end of a reaction
2. extremely efficient
3. high degree of specificity and usually specific to one type of substrate
4. denatured by heat and act most efficiently at optimal temperature
5. affected by pH and act most efficiently at optimal pH
6. activity regulated by inhibitors and activators

Question 6

Structure of enzymes

Answer 6

active site has specific 3D conformation and are globular proteins, when bonds are disrupted, enzyme is denatured

1. catalytic amino acids: R groups directly involved in catalytic activity once substrate is binded
2. binding amino acids: R groups form non-covalent bonds with substrate to hold it in position
3. structural amino acids: maintain sepcific 3D conformation of active site and enzyme as a whole
4. non-essential amino acids: no specific function and if replaced or removed, enzyme will not lose any of its catalytic function

Question 7

Define Cofactors

Answer 7

non-protein component that enzyme interacts with via covalent bonds or weak interaction in order to function as cofactors ensure substrate is correctly fitted into active site of enzyme

Question 8

Types of cofactors + their functioning mechanism

Answer 8

Inorganic metal ions:
1. mostly small divalent ions
2. component of active site or affect enzyme activity through **allosteric regulation **
3. Allosteric enzymes: multiple subunits and through conformational changes, bind activators or inhibitors at **site other than active site **
4. inorganic metal ions **bind reversibly **to enzyme and act by altering active site/allosteric sites to facilitate catalytic reaction carried out by the enzyme

Coenzyme:
1. loosely associate with enzyme and acts as a transient carrier of specific functional groups, hydrogen and electrons
2. enzyme that is catalytically active with its coenzyme and/or inorganic metal ion is known as holoenzyme, just the protein component is known as the apoenzyme

Prosthetic group:
1. prosthetic group binds tightly and pernamently to enzyme

Question 9

Define effective collision

Answer 9

when substrate and enzyme collide in the correct orientation for the binding of the subtrate to the active site of the enzyme

Question 10

Formation of enzyme-substrate complex

Answer 10

1. subtrate molecule held in active site by non-covalent bonds such as ionic and hydrogen bonds between R-groups of binding amino acids and substrate molecule
2. R-groups of catalytic amino acids catalyses the conversion of substrate to product
3. alteration of chemical conformation results in release of product from the active site as it is no longer complementary to the structure of the active site
4. enzyme is free for binding to other subtrate molecules and can be reused

Question 11

How enzyme lowers activation energy

Answer 11

1. orientating substrate, ensuring it is in correct orientation for reaction to take place
2. strains critical chemical bonds in substrate, allowing it to attain unstabe transition state
3. provides microenvironment that favours the reaction

Question 12

Specificity of enzyme

Answer 12

physical conformation and chemical properites due to R-groups of binding amino acids allows only subtrate molecules with complementary physical and chemical fit to enter active site to undergo catalysis

Question 13

Lock and key hypothesis

Answer 13

there is a complementary shape and/or conformation between the active site of the enzyme and subtrate molecule

active site of enzyme has a rigid structure and only subtrate exactly complementary to conformation of active site of enzyme can bind to active site of enzyme and undergo catalysis where reactant is converted to product

Question 14

Induced fit hypothesis

Answer 14

ground specificity where enzyme can catalyse reactions for a variety of substrates with similar physical and chemical properties

enzyme is:
1. active site does not have a rigid conformation that is can only bind to one type of substrate
2. active site has flexible conformation that can bind to more than one type of substrate
3. enzyme does not have precisely complementary conformation to substrate before binding

Interaction between enzyme and substrate: conformation of active site of enzyme changed slightly to bind substrate more tightly so R-groups of catalytic amino acids:
1. moulded to specific conformation
2. brought into closer proximity to chemical bonds in substrate for catalysis where substrate converted to products

Question 15

Rate of enzymatic reaction

Answer 15

amount of reactant converted to product by enzyme per unit time

measured by:
1. amount of product formed per unit time
2. amount of reactant used per unit time

Initial rate of reaction: initial amount of product formed/reactant used ÷ time duration

Question 16

Principal of limiting factor

Answer 16

in a reaction affected by many factors, factor shortest in supply in the limiting factor

1. rate of biological process consisting a series of chemical reactions, is limited by the slowest chemical reaction in the series
2. rate of biological process affected by many factors is limited by the factor shortest in supply, the limiting factor
3. increasing supply of limiting factor increases rate of reaction

Question 17

Factors affecting rate of enzymatic reaction

Answer 17

Concentration of substrate:
1. At low concentrations, not all active sites are occupied
2. substrate concentration is the limiting factor
3. increase substrate concentration, increase frequency of effective collisions between substrate molecule and enzyme active site
4. increase number of enzyme substrate complex formed per unit time and conversely amount of product formed per unit time
5. increase rate of reaction

1. at high concentrations, all active sites of enzymes are occupied
2. newly added substrate molecule has to wait for exisisting ESC dissociate to release product and free enzyme to form new ESC
3. increase in substrate concentration does not increase ROR
4. substrate concentration no longer limiting factor
5. increase enzyme concentration increases ROR

Concentration of enzyme
1. at low concentration of enzyme, increase in enzyme concentration
2. increase number of active site available
3. increase frequency of effective collisions between enzyme and substrate
4. increase ESC formed per unit time and coversely amount of product formed per unit time
5. increase ROR

1. at high concentration of enzyme, not enough subtrate molecules competing for active sites
2. enzyme concentration no longer limiting factor
3. substrate concentration becomes limiting factor

Temperature
1. at low temperatures, enzymes are inactive
2. increase temperature, increase KE
3. increase frequency of Ef
4. increase ESC formed per unit time and conversely amt of product formed per unit time
5. increased ROR until optimal temperature is reached
6. at optimal temperature, highest rate of ESC formation

1. temperature increases beyond optimal temperature, thermal agitation occurs
2. ionic, hydrgoen bonds and other non-covalent interactions stabilising specific 3D conformation of enzyme disrupted
3. loss in 3D conformation of active site of enzyme means enzyme active site is no longer a complementary fit with substrate and is denatured, loss of catalytic function
4. frequnce of Ef decreases
5. decrease in ESC formed per unit time and conversely amt of product formed per unit time
6. decrease in ROR

dual effect of temperature:
1. increases rate of formation of ESC
2. decreases stability of enzyme

pH:
1. at optimal pH, intramolecular bonds stabilising 3D conformation of enzyme intact
2. conformation of active site of enzyme most ideal for binding
3. highest frequence of Ef
4. highest rate of formation of ESC per unit time and amount of product formed per unit time
5. when pH slightly below/above optimal pH, there is a decline in rate of reaction due to alteration of 3D conformation of enzyme and subsequent decrease in affinity of enzyme for substrate

at low pH: more H+ ions, more negative charges of R-groups of amino acids neutralised
at high pH: less H+ ions, less negative charged of R-groups of amino acids neutralised
1. changes in ionisation of R-groups of amino acids disrupts ionic bonds and hydrogen bonds maintaining 3D structure of enzyme, denaturing enzyme
2. Structural amino acids:
- specific 3D conformation of active site no longer a complementary fit with substrate molecule
- no ESC formed
3. Binding amino acids: can no longer hold substrate in correct orrientation for catalytic reaction to occur
4. Catalytic amino acids: R-groups of catalytic amino acids no longer have correct ionisation/charge to catalyse catalytic reaction where reactant converted to product

rate of reaction affected by change in pH can be restored by returning pH back to optimal pH within limits, effects of changes in pH are reversible within limits

Question 18

Turnover number(Kcat)

Answer 18

maximum number of substrate molecules converted by enzyme to product per catalytic site per unit time

Question 19

Vmax

Question 20

Km(Michaelis Constant)

Question 21

Types of inhibition + mechanisms + graph observations and explanations

Answer 21

Competitive inhibitor:
Mechanism:
1. structurally similar to the substrate molecule and competes with substrate with active site
2. binds to active site and prevents substrate binding to active site from occuring

Graph:
1. Initial rate of reaction is lower in presence of competitive inhibitor
2. When [S] is very high, same Vmax is attainahle
3. However, longer period of time to produce same total amount of of product

Explanation:
1. Increase in [S] reduces effect of inhibition
2. Substrate and inhibitor in direct competition for enzyme active site
3. the greater the proportion of substrate molecules
4. the greater the chances of substrate out-competing inhibitor for active site
5. Rate of reaction can reach similar to Vamx of reaction without inhibitor
6. Final amount of product formed same as reaction without inihbitor as reactants can still be converted to products by other enzymes that are unaffected by the inhibitor

Non-competitive inhibitor
Mechanism:
1. bears no structural resemblance to the substrate
2. does not compete with substrate for enzyme active site
3. binds to another region of enzyme that is not the active site
4. alters the specific 3D conformation of the active site due to bond formation between enzyme and inhibitor
5. conformation of active site no longer a complementary fit to substrate
6. substrate cannot bind to enzyme and ESC cannot form

Graph:
1. initial rate of reaction lower in presense of non-competitive inhibitor
2. even when [S] is increased, initial rate of reaction in presence of inhibitor cannot reach same Vmax as reaction that is uninhibited

Explanation:
1. inhibitor binds to other site other than the active and forms bonds with the enzyme
2. alters the 3D conformation of the enzyme molecule, and conformation of active site no longer complementary fit to the substrate molecule
3. renders certain proportion of the enzyme molecules inactive, lowering Vmax
4. Substrate and enzyme not in direct competition for the same site, hence an increase in [S] hence no effect on the inhibition
5. Km remains the same as binding affinity of normal enzymes for substrate molecules remains the same
6. Total final amount of product formed the same as substrate molecules can be converted to products by enzymes unaffected by the inhibitor

Question 22

Similarities and differences of competitive and non-competitive inhibition

5

Answer 22

Similarities:
1. initial rate of reaction lowered
2. total amount of product produced remains the same

Differences:
Structure:
1. Competitive: resembles substrate
2. Non-competitive: does not resemble substrate
Binding site:
1. Competitive: complementary to and binds to enzyme active site
2. Non-competitive: not complementary to enzyme active site and binds to region other than active site
Effect of increasing [S]:
1. Competitive: Inhibition can be reversed as substrate can out-compete inhibitor for active site of enzyme
2. Non-competitive: no effect
Vmax:
1. Competitive: when [S] is very high, inhbited reaction can still attain Vmax
2. Non-competitive: Vmax is lower than uninhbited reaction
Km:
1. Competitive: affinity reduced(Km larger) as enzyme choose to bind to inhibitor instead
2. Non-competitive: the same as affinity of normal enzymes remains unchanged

Question 23

Allosteric regulation: definition + mechanism + cooperativity

Answer 23

Allosteric regulation: regulation of enzyme activity by binding a molecule at an allosteric site, these molecules are known as regulators(activators/inhbitors)

Mechanism:
1. Allosteric enzymes have 2 forms - active and inactive
2. Allosteric enzymes usually consist of 2 or more polypeptide chains and are multi-subunit enzymes
3. Each subunit has its own active site and allosteric site is usually where the subunits are joined

Allosteric activation: Activator binds to allosteric site, stabilising active form of enzyme increasing its affinity for its substrate

Allosteric inhibition: Inhibitor binds to allosteric site, stabilising inactive forms of enzyme, decreasing its affinity for its substrate

Rate of reaction of allosteric enzyme is sigmoidal in sahpe:
1. binding of an activator to an active site induces a favourable conformation change in active site of all subunits of enzyme
2. significantly amplifies response of enzyme to substrate -> amplication leads to sudden steep rise in rate of reaction

Cooperativity:
1. binding of one substrate molecule to one active site of multimeric enzyme trigger same favourable conformational change in active site of rest of subunits
2. binding of one molecule of substrate primes enzyme to accept additional substrate molecules

Question 24

Reversible and irreversible inhibition

Answer 24

Irreversible:
1. inhibitor loseely binds to enzyme via weak non-covalent bonds such as ionic bonds and hydrophobic interactions
2. effect of inhibitor is temporary, and can be easily removed from enzyme as it does not cause pernament damage to the enzyme
3. once inhibitor removed, catalytic function of enzyme is removed

Reversible:
1. inhbitor binds to enzymes by covalent bonds
2. causes pernament damage to the enzyme so it is unable to carry out catalytic activity

Question 25

End-product inhibition

Answer 25

when end-product of metabolic pathway accumulates, it acts an inhbitor and on the enzyme control preceding steps in the pathway inhibits by: 1. altering 3D conformation of active site of enzyme and decreasing its affinity for substrates 2. blocking entry of substrate to active site

Question 26

Control and regulation of metabolic pathways + advantages

Answer 26

Metabolism consists of thousands of chemical reactions arranged as intersecting pathways, need a structured system of contro provided by enzymes Advantages: 1. Biochemical reactions can proceed with no accumulation of products as product become substrate for subsequent reactions in metabolic pathway 2. Reactants can be modified in series of small steps: - release of energy to be controlled - minor modifications to be made to structure of the molecule - coupling of exergonic to endergonic reactions 3. each reaction in the metabolic pathway is catalysed by a specific enzyme allowing metabolism to be regulated and controlled as each enzyme becomes a point of control in the pathyway. enzymes regulated by activators/inhibitors, allowing for a finely balanced proportioning of metabolites among different pathways 4. spatial arrangement of steps in metabolic pathway such that product of one reaction is ideally located to be substrate of next enzyme. this allows for high local concentration of substrate and biochemical reactions can proceed rapidly 5. multi-enzyme complex is made of a team of enzymes that catalyse several steps in a metabolic pathway. arrangement is order of the sequence of reactions, such that product from first enzyme is substrate of enzyme adjacent to it, increasing efficiency of metabolic pathway

Question 27

Define Metabolism

Answer 27

totality of chemical reactions in an organism

Question 28

Location of enzymes

Answer 28

requires compartmentalisation as different enzymes have different reaction conditions Intracellular enzymes: 1. cytosol/nuclues 2. attached to cell surface membrane 3. in membrane bound organelles 4. attached to membrane of organelles Extracellular enzymes: synthesised within cell but packaged to be secreted out of cell