Enzymes

Question 1

What are enzymes

Answer 1

• biological catalyst by lowering activation energy
• globular protein (tertiary structure) with specific 3D conformation, active site with specific shape and charge
• intracellular (made by free ribosomes) and extracellular (synthesised at rough endoplasmic reticulum)

Question 2

Properties of enzymes

Answer 2

Chemically unaltered at end of reaction, effective in small amounts, can be reused

High turnover number (number of substrate molecules molecules converted into produces by 1 enzyme)

Globular protein with tertiary structure = soluble in aq env of cytoplasm/organelles/extracellular regions

Specific active site ; specificity due too shape/configuration and charge of active site

Question 3

Factors affecting enzyme activity

Answer 3

1. Temperature
2. pH
3. Substrate concentration
4. Enzyme concentration
5. Inhibitors

Question 4

How to explain Mode of Action of enzymes

Answer 4

1. Active site
2. Enzyme-substrate complex
3. Lowering Ea
4. Enzyme specificity (lock and key + induced fit hypothesis)

Question 5

Mode of action of enzymes : active site

Answer 5

Enzyme is a globular protein, far larger than substrate
Active site : 3-12 amino acid residues - contact and catalytic residues

Contact residues: contribute to enzyme specificity, form complementary shape of active site + have complementary charges to substrate

Catalytic residues: contribute to enzyme’s ability to catalyse biochemical reaction, increase activity of substrate by altering substrate’s charges

Other amino acids (majority): maintain globular shape = maintain active site shape; some may form other binding sites for inhibitors to regulate enzyme activity

Question 6

Mode of action of enzymes: enzyme specificity

Answer 6

Highly specific to substrates and reactions the catalyse

Enzyme + substrate = enzyme-substrate complex (weak hydrogen and ionic bonds) through effective collisions = enzyme + product

1. Lock and key hypothesis
   - suggests substrate is completely complementary in shape to active site, ie no change in shape when substrate binds
   - explains how enzymes area specific to substrate
2. Induced-fit hypothesis
   - suggests initial shape is not entirely complementary
   - substrate binding causes slight conformational change = fit more snugly
   - explains how enzymes lower Ea of reaction

Question 7

Mode of action of enzymes : lowering activation energy

Answer 7

Ea: energy needed to bring 1mole of substance to transition state at particular temperature; represent energy barrier
Increase Ea = decrease rate of reaction

Formation of enzyme-substrate complex = decrease Ea of reaction
- R groups of catalytic residues at active site can change the charge on substrate, alter electron distribution within substrate bonds = increase reactivity of substrate
- different substrate molecules in active site of enzymes are forced together in correct orientation for reaction to occur = bond formation between substrate molecule
- when complex is formed, certain bonds in substrate may face physical stress = increase likelihood of bonds breaking (induced fit hypothesis)

Question 8

Factors affecting rate of enzymatic reaction : enzyme concentration

Answer 8

Can greatly increase rate of reaction even at low concentration as they are chemically unchanged = can be reused

1. At low concentration
   - limiting factor, enzyme molecules are saturated and working at max rate
   - increase [enzyme] = more active sites = more effective collisions = more es complex formed = proportional increase in rate of reaction
2. At high concentration
   - [substrate] is limiting factor, insufficient substrate molecules to bind to empty active sites
   - increase [enzyme] does not increase rate of effective collisions/es complex formation = constant rate of reaction

Question 9

Factors affecting rate of reaction : substrate concentration

Answer 9

1. At low substrate concentration
   - [substrate] is limiting factor
   - increase [substrate] = more effective collisions since empty sites are avail = more es complex formed = proportional increase in rate of reaction
2. At high substrate concentration
   - [enzyme] is limiting factor, enzymes are fully saturated and working at max rate
   - increase [substrate] does not increase rate of effective collisions/es complex formation = constant rate of reaction

Question 10

Factors affecting rate of reaction : temperature

Answer 10

Temperature = amount of KE reactant molecules have

1. As temp increases to optimum
   - low temperatures near/below 0C = inactive enzyme, little KE for effective collisions
   - as temp increases, rate of reaction DOUBLES for EVERY 10C RISE (Q10 temperature coefficient 2 = rate at T+10/rate at T)
   - increase temp = increase KE of substrate and enzyme molecules = increase speed = more effective collisions = more es complex per unit time
2. At optimum temperature
   - maximum rate
3. Above optimum
   - rate decreases rapidly due to enzyme denaturation
   - large amt of KE = excessive vibration of AA residues in polypeptide chain of enzyme = ionic bonds, H bonds, hydrophobic interactions between R groups of AA disrupted = lose specific 3D conformation
   - active site shape affected= no effective collisions = no es complex formed ; enzyme is non-functional

• human enzymes: 35-40C
  Thermophilic bacteria enzymes : <70C = presence of disulfide bonds

Question 11

Factors affecting rate of reaction : pH

Answer 11

Large changes in pH causes enzyme to precipitate out

1. At optimum pH
   - rate of enzymatic reaction is maximum = effective collisions rate max = formation of es complex max
   - bonds maintaining specific 3D conformation are intact, shape of active site is complementary to substrate
2. At pH higher or lower than optimum
   - concentration of hydrogen ions is different from optimum
   - disrupts ionic bonds and hydrogen bonds between R groups of amino acids in enzyme = lose specific 3D conformation = active site shape affected
   - change in [H+ ions] = affect charges of active site and substrate = may no longer have opposite electrostatic charges
   - active site is no longer complementary in shape and charge to substrate - no effective collisions = no es complex formed

Question 12

Maximum rate of reaction (Vmax) and Michaelis-Menten constant (Km)

Answer 12

Vmax= max rate of an enzymatic reaction at a given [enzyme], temperature and pH in presence of excess substrate

Km= substrate concentration that enables enzymatic reaction to proceed at half its max rat (1/2 Vmax); constant for a particular enzyme, differs between enzymes

1/Km = affinity of enzyme for substrate
Bigger Km = decreased affinity for substrate = decreased rate of reaction

Question 13

Inhibition of enzymatic activity

Answer 13

Inhibitor : substance that prevents enzymes from catalysing its reaction = reduces rate of reaction through forming an enzyme-inhibitor complex

Reversible: binds to enzyme by weak interactions (eg hydrogen bonds), dissociates from enzyme rapidly, removal of inhibitor from enzyme restores function
- competitive, non-competitive, allosteric, end-product

Irreversible: binds to enzymes tightly via covalent bonds (slow dissociation), permanent
- heavy metal ions (Hg2+, Ag+), chemicals (arsenic)

Question 14

Inhibition of enzymatic activity : reversible - competitive inhibition

Answer 14

Close structural resemblance to substrate, shape is complementary to active site of enzyme
Competes w substrate -> increase [substrate] = increase probability of es complex formation = decrease effect of inhibitor

1. At low substrate concentration
   - probability of inhibitor binding > P substrate binding = active sites of enzyme blocked
   - lower frequency of effective collisions = lower rate of formation of es complex = lower rate of reaction
2. Increasing substrate concentration
   - more substrate molecules to compete = increase rate of effective collisions = increase formation of es complex = increase rate of reaction
3. At very high substrate concentration
   - substrate out-competes inhibitor to bind to active site
   - majority of active sites are bound by substrate molecules to form es complex
   - rate of reaction = Vmax as competitive inhibition is overcome by high substrate concentration

Km of enzyme increased, Vmax is the same but requires much higher [substrate]

Question 15

Inhibition of enzymatic activity : reversible - non competitive inhibition

Answer 15

No structural resemblance to substrate, binds to enzyme at another site (not active site)
Binding results in conformation change of enzyme = configuration of active site change = enzyme non-functional
At any given time, proportion of enzyme is bound by non-competitive inhibitor = non-functional

Decreases effective enzyme concentration
- increasing [substrate] has no effect

Km of enzyme not affected, Vmax is lowered

Question 16

Inhibition of enzymatic activity : reversible - allosteric (what is an allosteric enzyme)

Answer 16

Type of inhibition specific for allosteric enzymes: consists of more than one polypeptide chain (quaternary structure)
- each polypeptide chain makes up one subunit of enzyme, each subunit has its own active site
- allosteric sites: where polypeptides join
- conformation alternates between high affinity active form and low affinity inactive form -> regulated by substrate binding / biding of allosteric activators or inhibitors

Question 17

Inhibition of enzymatic activity : reversible - allosteric (substrate binding and cooperativity)

Answer 17

Co-operativity: substrate binding to one active site of one subunit = conformation of whole enzyme to active form = substrate biding to other active sites in other subunits

Enzyme kinetics graph is sigmoidal (S-shaped)

Question 18

Inhibition of enzymatic activity : reversible - allosteric (activators and inhibitors)

Answer 18

Allosteric activators and inhibitors bind to allosteric sites of allosteric enzymes

Activator: stabilises active form of enzyme
Inhibitor: stabilises inactive form of enzyme

Thus allosteric inhibition = non-competitive as inhibitor binds to site other than active site, which changes active site shape to be less complementary to substrate

Eg effect of ATP on phosphofructokinase (involved in glycolysis- cellular respiration)
- when cell has high [ATP], ATP acts as allosteric inhibitor, binds to allosteric site of phosphofructokinase to inactivate enzyme
- thus glucose can be used in other processes

Question 19

Inhibition of enzymatic activity : reversible - end-product inhibition

Answer 19

Metabolic pathway involves series of reactions catalyse by enzymes : allows for greater level of regulation as pathway is controlled at each enzyme

End-product of pathway accumulates = acts as inhibitor of enzyme in pathway = stop synthesis of end-product (FEEDBACK INHIBITION)

Prevents wastage of resources
Allows intermediates in pathway to be channeled to another pathway to produce other end-products
Prevents potential adverse effects due to accumulation of end-product

Question 20

Why do metabolic pathways occur as series of steps rather than just 1 or 2? (End product inhibition)

Answer 20

1. Feedback inhibition by end-product
2. Greater level of regulation as pathway is controlled at each enzyme
3. Intermediates from one pathway can become substrates for other pathways
4. Smaller release of energy in each step (vs sudden large release of energy) = better utilisation of energy + prevents cell damage