B1-4 Enzymes

Question 1

Definition of Enzymes

Answer 1

Biological catalysts that possess the ability to speed up a reaction without being themselves changed at the end of a reaction

Question 2

How do enzymes work (not the hypotheses ah don’t think so far)

Answer 2

Enzymes increase the rates of chemical reactions by lowering the free energy barrier that separates the reactants and products i.e. activation energy

Question 3

Definition of Activation energy (Ea)

Answer 3

Minimum amount of energy required to start a chemical reaction

Question 4

Lowering of Ea

Answer 4

At physiological temperatures, few molecules can overcome Ea barrier and can only do so with the use of a catalyst

Activation energy is lowered by enzyme
More reactant molecules can surmount the energy barrier to reach the transition state to be converted into product molecules
Total energy difference/ free energy change/ Gibbs free energy change between reactant and product molecules remains the same

Question 5

Properties of Enzymes (6)

Answer 5

Effective in small amounts (chemically unaltered, reusable)
Extremely efficient
High degree of specificity
Denatured by heat and act most efficiently at optimum T
Affected by pH and act most efficiently at optimum pH
Activity can be regulated by activators and inhibitors

Question 6

Structure of enzymes

Answer 6

Most are globular proteins
Have specific 3D conformation necessary for their action
3D structure must be maintained for enzyme to remain functional
An enzyme can be denatured when bonds holding them in specific 3D conformation are disrupted

Question 7

4 categories of aa residues

Answer 7

Catalytic aa residues - R groups involved in catalytic activity
Binding aa residues - R groups hold substrate in position via covalent bonds
Structural aa residues - Maintain specific 3D conformation of active site and enzyme as a whole
Non-essential aa residues - No specific function, can be removed or replaced without loss of enzyme’s catalytic function

Question 8

Cofactor - Inorganic metal ions

Answer 8

Usually small and divalent (charge +2)
Component of active site or allosteric regulator
Bind reversibly to enzyme and act by altering enzyme’s active and/or allosteric sites to facilitate catalytic reaction carried out by enzyme

Question 9

Cofactor - Coenzyme

Answer 9

Loosely associated with enzyme during reaction
Act as transient carriers of specific functional groups, hydrogens, or electrons
Most are derived from vitamins

Question 10

Cofactor - Prosthetic Group

Answer 10

Tightly bound to enzyme on permanent basis

Question 11

Allosteric Enzymes

Answer 11

Alternate between active and inactive form
Have multiple subunits and through conformational changes, binds activators of inhibitors at sites other than active site

Question 12

How Enzymes lower Ea

Answer 12

Orientate S in close proximity, in correct orientation, to undergo chemical reactions
Strain critical bonds in S molecule(s), allowing S to attain unstable transition state
Provide microenvironment that favours reactions

Question 13

Lock and Key Hypothesis

Answer 13

There is an exact fit/complementary shape or conformation between S and active site of E
E is viewed as a rigid structure, where only S exactly complementary to conformation of active site are able to bind to active site for catalysis
Explains substrate specificity of enzymes

Question 14

Induced Fit Hypothesis

Answer 14

Active site conformation is not precisely complementary to that of S before binding
Active site is not a rigid conformation that fits only one type of substrate

Upon binding, active site of E changes conformation slightly to bind S even more firmly so that R groups of catalytic aas at active site are moulded into specific conformation and brought into close proximity to the chemical bonds in S to facilitate catalysis

Further explains group specificity where one enzyme is able to catalyse reactions for a variety of substrates that share similar structural or chemical properties

Question 15

Rate of enzyme-catalysed reaction and how to measure

Answer 15

Rate is the amount of S which is converted to P by enzymes per unit time

Measured by rate of product formation or substrate usage

Question 16

Competitive Inhibitor

Answer 16

Structurally similar to S, compete with S for binding to active site
Although it is not acted upon by E, it remains bound to the active site and prevents S binding to active site

Initial rate reduced, but both reactions reach same Vmax
Requires longer time in presence of inhibitor to reach same Vmax
Km higher for inhibited

Question 17

Why increase in S conc reduces effect of competitive inhibition

Answer 17

Because S and inhibitor are in direct competition for E’s active sites
Greater proportion of S - Greater chances of out-competing inhibitor - Rate of reaction almost equivalent to Vmax can be attained
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor

Question 18

Non-competitive Inhibitor

Answer 18

Bears no structural resemblance to substrate, does not compete with S for active site

Binds to part of E that is not active site
Binding of inhibitor alters 3D conformation of E and active site
E molecule no longer has active site complementary in conformation to S
Hence S does not bind to E active site and no E-S complex can be formed

Question 19

Why increase in S conc reduces effect of non-competitive inhibition

Answer 19

Even when substrate concentration is very high, initial rate of reaction does not reach same Vmax as uninhibited reaction
Binding of non-con to site other than active site causes change in 3D conformation of enzyme active site that prevents S from binding

Question 20

Why non-competitive inhibitor has lower Vmax

Answer 20

Certain proportion of E molecules are rendered inactive
As S and inhibitor are not in direct competition for same site, increase in S concentration has no effect on inhibition
Km remains unchanged as affinity of enzyme for substrate remains unaffected
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor

Question 21

Why non-competitive inhibitor has lower Vmax

Answer 21

Certain proportion of E molecules are rendered inactive
As S and inhibitor are not in direct competition for same site, increase in S concentration has no effect on inhibition
Km remains unchanged as affinity of enzyme for substrate remains unaffected
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor

Question 22

Allosteric Regulation

Answer 22

Regulation of an E by binding of molecules (regulators: activators, inhibitors) at an allosteric site i.e. site other than active site

Question 23

Allosteric enzyme structure

Answer 23

Most allosterically regulated E are composed of 2 or more polypeptide chains - Multi-subunit E
Each subunit has its own active site and allosteric sites are usually located where subunits are joined

Question 24

Allosteric Inhibition

Answer 24

Binding of activator to active site induces favourable conformation change in active site of all subunits of enzyme
This significantly amplifies response of E to substrates. The amplification results in sudden steep rise in rate of reaction

Question 25

Cooperativity

Answer 25

Binding of 1 S molecule to an active site of a multimeric enzyme triggers same favourable conformation change in active site of all other subunits of E
Likewise amplifies response of E to S - 1 S molecule primes E to accept additional S molecules

Question 26

Reversible vs Irreversible inhibition

Answer 26

Inhibitor binds via weak non-covalent (hydrogen bonds, hydrophobic interactions) VS covalent bonds
Reversible - Effect is temporary, inhibitor can be easily removed and causes no permanent damage
Irreversible - Inhibitor causes permanent damage to E as it is unable to carry out catalytic activity

Question 27

Factors affecting Rate of Enzymatic reaction

Answer 27

Substrate Concentration
Enzyme Concentration
Temperature
pH

Question 28

Kcat / Turnover number

Answer 28

Maximum number of molecules of S that an enzyme can convert to P per catalytic site per unit time

Question 29

General steps to explaining rate of reaction

Answer 29

Frequency of effective collisions
Rate of formation of enzyme-substrate complexes
Rate of product formation

Question 30

Effect of low T

Answer 30

Enzymes are inactivated

Question 31

Effect of T beyond optimum T

Answer 31

Decrease in rate of reaction despite increasing frequency of collisions (decreasing stability)
Thermal agitation of E molecule disrupts hydrogen bonds, ionic bonds, and other non-covalent interactions that stabilise the specific 3D conformation of protein molecule

Loss in 3D conformation of E and that of its active site so that there is no longer a complementary fit with S
E said to be denatured and lose its catalytic function

Frequency of effective collisions decreases… (typical stuff)

Question 32

Changes in pH can affect E activity by…

Answer 32

Altering ionic charge of acidic and basic R groups

Low pH - More H+ ions available to neutralise negative charges in enzyme
Higher pH - Less H+ ions available to…
Change in ionisation of aas disrupts ionic bonds / hydrogen bonds that maintain 3D conformation of enzyme, denaturing enzyme

Care about effect on all aa residues except non-essential ones

Question 33

Optimum pH

Answer 33

Enzymes work most efficiently
Rate of reaction at max, intra-molecular bonds are intact and conformation of active site is ideal for binding, therefore frequency of EC is highest with largest amount of ES complexes formed

At pH values only slightly above and below optimum, many enzymes will have marked decline in E activity as 3D conformation is altered and affinity for substance is correspondingly decreased

Optimum pH is usually pH of environment in which the enzyme normally functions

Question 34

Vmax

Answer 34

Max rate at which E is able to perform the reaction in a specific concentration of E and excess S

Question 35

Km

Answer 35

S concentration that allows an enzyme-catalysed reaction to proceed at half of Vmax
Represents affinity of E for particular S
Low value + High affinity

Question 36

Feedback / end-product inhibition

Answer 36

Cellular control mechanism in which an enzyme’s activity is inhibited by the enzyme’s end product