35. Proteins and enzymes

Question 1

what are proteins?

Answer 1

• most important and ubiquitous macromolecules in cell
  – all antibodies
  – most enzymes and hormones
  – connective tissue
  – muscle fibrils
  – cillia and flagella
• almost all cellular functions

Question 2

what is the structure of proteins?

Answer 2

• linear polymers of amino acids
  – joined in genetically determined sequence
• synthesised by ribosomes
  – under genetic direction of mRNA
• properties depend on:
  – composition of AA sequence
  – post-translational modifications (folding, subunit joining, glycosylation) a

Question 3

what are amino acids?

Answer 3

• 20 standard types of AAs
  – 10 considered essential
• most proteins contain all 20
  – proportions vary

Question 4

what is the basic structure of amino acids?

Answer 4

• carboxyl groups
• amino group
• hydrogen atom
• R-group
  – organic substituent (side-chain)
• all attached to C atom

Question 5

how are amino acids characterised?

Answer 5

• chiral molecules
  – except glycine (R=H) all have asymmetric C
  – exist in 2 stereoisomeric forms (D/L-isomers)
• both isomers exist
  – L-amino acids incorperated into proteins
• characteristic depend on R-group
  – range from simple (H)
  – to complex aromatic structures

Question 6

what is aspartame?

Answer 6

• artificial (non-saccharide) low calorie sweetener
  – L-phenylalanyl-L-aspartyl-methyl ester
• widely used in soft drinks and confectionary products
• approx. 200x sweeter than sucrose
  – only 4 calories/g

Question 7

what is the structure of aspartame?

Answer 7

• dipeptide
  – composed of 2 amino acids
• L-phenylalanine (Phe)
  – as methyl ester
• L-aspartic acid (Asp)

Question 8

what are the structures and properties of amino acids?

Answer 8

• 9 AA’s non polar
  – hydrophobic
  – no O or N in R-group
  – typical interior of protein (3-D structure)
  – if protein mainly hydrophobic AA, will seek hydrophobic regions (interior membrane)
• 11 hydrophilic
  – R-group disticntly polar
  – AAs tend to cluster at protein surface

Question 9

what is the peptide bond?

Answer 9

• during protein synthesis
  – each AA attached to growing chain in condensation reaction
  – bond between AA ususally peptide bond
• nature of bond gives protein intrinsic diretionality
  – N-terminus and C-terminus

Question 10

what is the peptide bond formation?

Answer 10

• mRNA and tRNA (genetically encode)
• requires energy to acivate incoming AA
• initial polypeptide not functional protein
  – functional protein has unique stable 3-D shape

Question 11

what are the levels of organisation in protein structures?**

Answer 11

–

Question 12

what is the primary structure of proteins?

Answer 12

• amino acids
  – in polypeptide chain
• peptide bond
  – between each amino acid
• sequence of AA
  – determined by DNA sequence
• not a functioning protein

Question 13

what is the secondary structure of proteins?

Answer 13

• hydrogen bonds between AA
• polypeptide chain coils (folds) into
  – alpha-helix
  – B-sheet

Question 14

what is the tertiary structure of proteins?

Answer 14

• 3D folding single polypeptide
• hydrogen bonds
• di-sulfide bonds
• electrostatic interactions
• hydrophobic effect

Question 15

what is the quaternary structure of proteins?

Answer 15

• association of two or more polypeptides
  – form multimeric protein

Question 16

what is the denaturation fo proteins?

Answer 16

• loss of higher order structure
• associated with loss of biological function
  – function not associated with first degree structure
• caused by exposure to:
  – extreme heat
  – extreme pH
  – certain chemicals
• peptide bonds (covalent) unaffected
• depending on severity of denaturant
  – protein may/not refold to original conformation

Question 17

how is thermal denaturation applied in industry?

Answer 17

• Taq adapted to live at high T
• extract genomic DNA
• isolate portion of DNA from Taq
  – that encodes Taq DNA polymerase
• insert into E.coli

Question 18

how are proteins secreted?

Answer 18

• synthesised with 15-30 peptides at N-terminus
• signal sequence/peptide
  – rich in hydrophobic AAs
• ribosomes synthesising secretory proteins
  – often attached to cell membran
• signal sequence cleaved off by signal petidase
  – after secretion to produce mature protein

Question 19

what is the protein 3D structure and how is it dictated?

Answer 19

• conformation
• dictated by:
  – interactions with solvent
  – pH and ionic composition of solvent
  – sequence of protein (dictated by DNA sequence of gene encoding protein)

Question 20

what are functional proteins?

Answer 20

• eg. enzymes
  – protein catalysts
• highly specific for reaction they catalyse
  – and molecules acted upon (substrate) and produced

Question 21

what affects functional proteins?

Answer 21

• substrate
• conditions
  – pH
  – T
• inhibitors
• presence/absence of inhibitor or required co-factors

Question 22

what are industrial enzymes?

Answer 22

• textile processing
• grain processing
• food processing
• cleaning
• feed enzymes
• diagnostic/pharma
• waste management
• other

Question 23

what are enzymes?

Answer 23

• catalysts
• usually proteins
  – some ribonucleic acid (RNA) molecules
• perform ciritcal lowering of reaction’s activation energy
  – amount of energy required for reaction
• don’t change reactions delta G value
  – don’t affect free energy of reactants/products
  – don’t change whether reaction energy is releasing/absorbing overall

Question 24

how do enzymes affect activation energy?

Answer 24

• lower Ea
• bind to reactant molecule in such a way that chemical bond-breaking/forming takes plac emore readily
• increase reaction rate

Question 25

how do enzymes work as biocatalysts?

Answer 25

• allow reactions to occue at mild conditons
  – T for living organisms
  – dilute aqueous solutions
  – biological pH range
  – atmospheric pressure
• specificity and selectivity
  – stereospecific (act only one stereoisomer of substrate)
  – reaction specific (no wasteful bi-products; 100% yield of desired product)

Question 26

what are the different enzyme classifications?**

Answer 26

-

Question 27

what are active sites & substrate specificity?

Answer 27

• catalyse reaction, enzyme will bind to one/more reactant molecule
  – enzyme’s substrates
• substrate broken down to multiple products/come together forming larger molecule
  – or swap pieces
• active site is where sibstrate binds