6 - Enzymes I: Introduction to enzymes

Question 1

Louis Pasteur

Answer 1

fermentation requires living cells (1850)
Live yeast + sugar = alcohol
Cell-free yeast extract + sugar = no alcohol

Question 2

Wilhelm Kühne

Answer 2

proposed the term “enzyme” (1878)

Question 3

Buchner brothers

Answer 3

fermentation in cell-free extracts from yeast (1897)

agreed cells contained a biological catalyst

Question 4

Biological catalysts; functions

Answer 4

• Regulate the flow of molecules through all metabolic
pathways
• Build structures of cells (and break them down again)
• Break down invading pathogens and parasites - E.g. lysozyme – tears, egg white etc

Question 5

Biological catalysts; uses

Answer 5

Lipases, proteases; stains - Cellulase; ‘bobbles’ - Low temp, less water
Fondant centres – invertase breaks down sucrose in the fondant centre and prevents it from crystallising
Rennet – calf
stomach; now GM microorganisms, Mixture – mainly Chymosin – curdles casein in milk
Cotton and cellulosic textile manufacture – stonewashed denim (cellulase), de-starching - amylase

Question 6

Biological catalysts

Answer 6

• Enzymes increase reaction rates without enzyme – thermodynamically unfavourable e.g. prolyl isomerase – cis to trans (x 1 million!)
• Enzymes remain unchanged after the reaction repeat again & again; a few may be ‘modified’ (later)
• Chemicals acted upon = substrates, end point = products
• Most - but not all - enzymes are proteins: ‘Ribozymes’ (ribonucleic acid enzymes) – ‘self-splicing’ RNA (removes introns

Question 7

Substrate-specificity

Answer 7

• Low specificity: Subtilisin - a bacterial protease that cleaves peptide bonds indiscriminately
• Moderate specificity: Trypsin - a digestive enzyme that cleaves peptide bonds on the carboxyl sides of Lys or Arg
• High specificity: Thrombin - a blood clotting enzyme that cleaves between Arg and Gly within specific amino acid sequence motifs

Question 8

Enzyme classification

Answer 8

• Most enzyme names end in “-ase”: ATPase (breaks down) ATPsynthase (synthesises)
• Exceptions are proteolytic enzymes that end with “-in” e.g. trypsin, pepsin

Question 9

Enzyme Commission (EC) numbers

Answer 9

International Union of Biochemistry and Molecular Biology :Enzyme Commission
EC numbers take the form : 1.2.3.4
• First digit from 6 major classes (bulk descriptor of what the enzyme does)
• Second and third digits give further details for the type of reaction (bonds, groups)
• Fourth digit gives substrate (general descriptor, e.g. amino acid, nucleic acid, lipid, etc)

Question 10

Measurements of enzyme activity

Answer 10

Activity of an enzyme indicated by rate of reaction: amount of product produced per unit time
• SI Unit - katal (kat) : The amount of an enzyme that converts 1 mol of substrate per second under standard* assay conditions – BIG numbers!
• International activity unit (U) : An amount of enzyme that will catalyse the transformation of 1 micromole of the substrate per minute under standard* assay conditions.

Question 10

Thermodynamics – Free energy

Answer 10

• Breaking bonds requires energy, forming bonds liberates energy However, even most exergonic reactions need an initial ‘spark’ of energy to kick start them (collision and bond destabilisation e.g. initial bonds in glucose and ATP opposite need to be broken)

Question 11

Exergonic reaction

Answer 11

Releases energy to surroundings

• Will occur spontaneously if there is enough ‘Activation energy’

Question 12

Endogonic reaction

Answer 12

reactions that require overall input of energy

Question 13

Thermodynamics – Activation energy

Answer 13

Initial input or ‘spark’ of energy = Gibbs free energy of activation (DG‡)

Question 14

Thermodynamics – Activation energy; energy profile diagram

Answer 14

• The difference between *free energy of the products and the free energy of the reactants is the Delta G (total reaction change in free energy)
• At the summit the molecules are at an unstable point, the transition state
• The difference between the free energy of the reactants and the transition state is the Delta G‡ (change in Gibbs free energy of activation)
Delta G‡ is limiting

Question 15

How do enzymes increase reaction rates?

Answer 15

• Enzyme speed reactions by lowering EA- The transition state can then be reached more easily because less energy is required to drive reaction forward

Question 16

The evolution of the enzyme-substrate complex hypothesis (metal stick)

Answer 16

• First step in catalysis is the formation of the enzyme-substrate complex, ES
Lot of charge interactions between AAs in enzyme and AAs substrate
• Active site is shaped like the transition state
• Binds to substrate then magnets start to pull middle of stick upwards to form transition state
• This pull of the magnets provides energy for the bending of the substrate
thereby reducing the extra (activation) energy (EA) required to form transition state