1. Biologic roles of Protein
    Structure, transport, enzymes, motility, immune system, metabolic regulation (hormones)
  2. Amino Acid Structure
    • H a-carbon surrounded by a carboxyl group (right), amino group (left), side chain (below)
    • -
    • H2N--C---COOH
    • -
    • R
  3. Hydrophobic aliphatic (non-polar) sidechains:
    • Glycine (G) Amanine (A) Valine (V) Leucine (L) (isoleucine (I), methionine (M), proline (P))
    • -H -CH3 -CH--2CH3 -CH2--CH--2CH3
  4. Hydrophobic aromatic (non-polar) side groups
    • phenylalanine (F) Tryptophan (W)
    • -CH2-benzyne ring -CH2-C-(2 rings w/ NH group)
  5. Hydrophilic charged (polar) side chain
    • Asparatate (D) Glutamate (E) Lysine (K)
    • -CH2-C-2O- -CH2-CH2-C-2O- -CH2-CH2-CH2-CH2-NH3+

    also: arginine (R), histidine (H) (+) charge
  6. Hydrophilic and neutral (polar) side chains
    • Asparagine (N) Glutamine (Q) Serine (S)
    • -CH2-C-O/NH2 -CH2-CH2-C-O/NH2 -CH2-OH

    Also: Threonine (T) and cysteine (C)
  7. Hydrophobic vs. hydrophilic R groups
    • Hydrophobic: hydrophobic interaction in aqueous environment on INSIDE of protein
    • Hydrophilic: H bonding with water in aqueous environment on OUTSIDE of protein
    • (charged side chains have electrostatic interactions)
  8. (Met) Amino Acid
    Met is always the 1st AA in sequence as protein synthesis starts- met is rarried by tRNA in the initiator site in the ribosomes. (may be removed post initiation)
  9. Modification of amino acids
    Glycosylation and Phosphorylation- 20 AA coded by DNA sequence but over 300 AA in the body
  10. pH effect on Amino Acids
    • Amino acids have an amino group (basic) or carboxyl group (acidic) which are ionized in solution depending on pH.
    • ex: glycine at pH7 has no net charge so at low pH (acid) it is positivly charged, at high pH (basic) it is neg. charged
  11. Isoelectric point (pI)
    • the pH value when no charge on amino acid or protein (equal positive and neg charge) will vary with each AA. depends on R group.
    • acidic side chaine will be neg at pH7- no charge at low pH
    • basic side chain wil be positive charge at pH7- no charge at hight pH
  12. Polymerisation (peptide bonds)
    bonds linking AA's in proteins are called peptide bonds. they are formed by carboxyl group of one AA linked to amino group of another AA. they form amide (peptide) bond. in the process one molecule of water is lost. needs energy.
  13. Amino acid residues
    each amino acid unit in peptide or protein
  14. lengths of aa chains
    • short peptides: oligopeptides (less than 50 residues)
    • polypeptides
    • proteins
  15. Unique AA sequence:
    determined by gene sequence
  16. Peptide bond qualities
    • peptide bond is rigid and planar.
    • H and O are always in trans config. (opposite each other).
    • peptide partial double bond- either side single bonds can rotate
    • bond length- 1.32A (somewhere beterrn double and single bond length)
    • sequence ALWAYS given from N terminal to C terminal (N is beginning of peptide chain)
  17. Primary Structure of Proteins
    Simple, involves only the amino acid sequence (linked by peptide bonds)
  18. secondary bonding of proteins
    • H bonding only in peptide backbone (R groups not directly involved)
    • protein adopting a specific conformation
    • Type of amino acid can influence the type of secondary structure a protein adopts
    • 2 main types (alpha helix and beta sheet)
  19. Alpha Helix
    • h bonds run parallel in direction of helix
    • AA 3-4 apart in sequence are close together in helix
    • amount of helix varies depending on protein
  20. Beta Sheet
    • involves H bonding between strands
    • ex: silk- antiparallel sheets
    • proteins fold into compact structure involving reversal direction of polypeptide chains
    • achieved thro beta turn
    • H bonding between CO and NH three residues apart in chain
    • (proline often found at beta turn)
  21. Super Secondary structure
    • Clusters of secondary structure
    • Beta-strand - alpha helix - beta strand (beta, alpha, beta unit)
    • can generate separate domains
    • domains may have different functions (depends on R group present)
    • (evolution- duplication/removal of domains within or between proteins)
  22. Special helix in collage
    • Rod shaped structure- NOT same as alpha helix
    • more open than alpha-helix
    • 3 amino acid redicues per turn (vs. 3.6)
    • conserved sequence (Gly-X-Y-Gly-X-Y) (X and Y are proline and hydroxyproline)
    • stabilised by steric repulsion from proline and hydroxyproline rings (keeps it twisted)
  23. Collagen triple helix
    • each strand forms a helix
    • 3 collagen strands entwine- triple helix
    • glycine occupies every 3rd position (small, so fits in interior)
    • with Proline and hydroxyproline to exterior
    • Stabilised by H bonds between CO and NH and OH of hydroxyproline (of different chains)
  24. Scurvy
    • Vitamin C deficiency (ascorbate)
    • vitamin C co-enzyme for prolyhydroxylase
    • insufficient hydroxylation of proline
    • fewer H bonds to hold collagen together--> unstable/weak/breaks in skin, bones, teeth, etc.
  25. Protein Tertiary structure
    • Side chain (R group) interactions
    • weak, non-covalent interactions but stabilize protein
    • spatial arrangement and interactions of AA's so far apart
    • responsible for folded, biologically active protein (3-D structure)
  26. Types of bonding in tertiary structures
    • H bonding (ser, thr OH groups)
    • Electrostatic interactions between charged groups (lys, arg, his, glu, asp)
    • hydrophobic interactions (leu, val, phe...etc)
    • Van der Waals' forces (short range/weak)
    • disulphide bonding between cysteine residues strong interaction
  27. Protein denaturation
    • Disrupts bonding--> insoluble
    • *Heat- breaks weak bonding (eg egg white)
    • *pH- affects H bonding and electrostatic interactions
    • detergents and organic solvents (hydrophobic bonds)
    • chaotropic agens- eg urea, guanidine HCL form H bonds with AAs and disrupt existing H bonding and hydrophobic interactions
    • *Most common biologicaly
  28. Quatinary structure
    • Complex of 2 or more separate polypeptide chains
    • interactions between subunits
    • non-covalen and covalent interactions
    • example: Haemoglobin- 4 subunits
  29. Importance of 3D structure for protein function:
    • Allows proteins to interact with other molecules
    • form bonds with other molecules
    • usually weak but specific interactions
    • why proteins act as enzymes, receptors, etc.
  30. Importance of 1y structure/denaturing agents
    • denaturing agents: Urea, guanidine HCL- disrupt H and hydrophobic bonds
    • mercaptoethanol- reducing agent reduces disulphide bonds (-S-S- --> - SH -SH)
  31. Denaturation of 3y structure
    • denatured proteins lose all enzume properties- for example:
    • Native ribonuclease denatured with urea, disulfide bonds fall apart but by removing urea and maercaptoethanol the bonds reform
    • so all information for complex 3D structure is contained in amino acid sequence
    • 1y sequence dictates structure
    • chemically- folding slow process
    • physiologically- protein folding involves chaperone proteins, scaffold, helps
  32. Importance of correct 1y structure
    • Gene mutations- changes in AA sequence
    • Substitutions- conservative (same type of AA), radical (different type of AA- example sickle cell anemia)
    • Change can affect ligand binding or enzyme activity can affect shape of protein (esp cysteine substitutions)
  33. Insertions or Deletions
    • Can cause loss of protein function- but not always (if remove non-essential part)
    • deletions/substitutions can determine binding site residues
  34. X-ray crystallography
    • best way to predict complete 3D structure of a protein.
    • need lot pure protein, readily forms crystals
    • bombard with x-rays- build up 3D image
  35. Protein environment (Aqueous Environment)
    eg water soluble protein
    • Hydrophilic amino acids outside (h bonding, electrostatic interactions)
    • Hydrophobic amino acids inside (hydrophobic interactions, Van der Waals' forces)
  36. Protein Environment: in hydrophobic environment
    (eg transmembrane protein)
    • Hydrophilic AAs on Aqueous side
    • Hydrophobi AAs in mimbrane lipid bilayer
  37. Fibrous proteins
    • Very Strong
    • usually structural proteins (eg collagen, keratin, actin, myosin, extracellular or intracellular)
    • extensive 2y structure, stabilised by disulphide and H bonds
    • undergo modifications- increase stability (eg collagen)
  38. Globular proteins
    • mostly non-structural proteins
    • compact shape
    • 3y structure important to fundtion (more folded)
    • ligand binding sites
    • eg myoglobin, haemoglobin, albumin, enzymes, receptor proteins
  39. Abnormal deposits of protein fibrils...
    • Alzheimer and prion disease
    • Alzheimer's- beta-amyloid protein forms abnormal extended beta-sheets
    • (globular-->fibrous and not converted back!)
    • Prion disease (scrapie, BSE, CWD, CJD)
    • cellular form (PrPC) is globular--> Scrapie form (PrPSC) is fibrous
  40. Theory of Amyloid diseases (globular/fibrous proteins)
    • Normal conditions: fibrous -----------<-> Globular (helped by chaperones
    • disease condition: fibrous<-->------------ Globular (not well known what the "switch" is)
    • genetic or old age?
    • Defect in maintaining globular form- protein destabilized to fibrous form- only dz that passes by proteins
    • transmissible (prions)- already formed fibrils act as template globular-->fibrous (rapid progression)
  41. Scrapie resistance breeding programme
    • April 2005- compulsory EU breeding programme
    • breed from sheep resistant to Scrapie- ARR type (1)
    • not from susceptible sheep- VRQ type (5)
    • (DEFRA web site)
    • 3 codons in the gene encoding PrPC- AA changes
    • Resistant scrapie form does not progpagate well
  42. 4y structure characteristics
    • Protein with >1 subunit
    • subunits interact
    • influence binding at each binding site
    • conformational changes when ligand binds
  43. Allosteric effects
    • Tense form (T)- low affinity for ligand
    • Relaxed form (R)- high affinity for ligand
    • ligand binds to T, increases affinity of other subunit
    • stable/less stable/more stable
    • T --> R -----> R
    • T<------T <-- R
  44. Regulation of Protein Activity (allosteric)
    • Allosteric regulators bind at other sites- alter affinity for ligand
    • Positive allosteric regulators increase ligand affinity
    • Negative allosteric regulators decrease ligand affinity
    • Protein/enzyme activity can be changed
    • regulated activity
    • physiologically important (controlled depending on body conditions)
    • ex: haemoglobin- allosteric protein with 4 binding sites
  45. Proteins VS. DNA
    • Proteins: structural/functional (collagen/enzymes), made up of amino acids, linked by peptide bonds, alpha helix, beta sheet, collagen triple helix
    • DNA: genetic material, made up of deoxyribonucleotides, linked by phosphodiester bonds, DNA double helix
  46. Protein Purification
    • Tissue- releases many proteins, salts etc.
    • seperate on basis of:
    • size (dialysis/ultrafiltration, gel filtration, gel electrophoresis)
    • charge (ion-exchange chromatography, isoelectric focussing)
    • Ligand binding (affinity chromatography specific interaction of proteins with other molecules)
  47. Size separation/ Protein purification
    • Dialysis: Semi-permeable membrane, small molecules pass out
    • Ultrafiltration: same principle, but small molecules are foced through membrane by pressure or centrifugation
    • Gel filtration: large molecules out 1st
    • Gel electrophoresis: through gel support, SDS-PAGE, SDS- binds to proteins and gives neg. charge, move to anode= migration depends on size (small molecules move rapidly/large ones at top, good resolution but low amount protein)
  48. Separation of protein by charge
    • Proteins with + net charge bind to - molecules and vise versa
    • separate in a column containing charged beads
    • isoelectric focusing (IEF): isoelectric point (pI) pH when protein has no net charge separate by electrophoresis (NO SDS) protein will move in pH gradient until pI stops when no charge
  49. Separation of Protein on size and Charge
    • isoelectric focusing
    • very good resolution in 2D
    • separate complex protin mixtures (eg tissue)
    • advanced technology- identify each spot
    • digest protein to peptide fragments
    • mass spectrometry
    • amino acid sequence
    • fully automated
    • detailed analysis- proteomics
  50. Proteomics
    • Study protein profile of cell or tissue
    • proteins present at particular time and in particular cell or tissue
    • progression from Genomics (genome study)
    • application: normal vs. cancer cell
    • qualitative (differences in AA sequence)
    • quantitative (increase or decrease in proteins
    • non-biased approach- look at all proteins
  51. Basic Enzyme catalysed reaction
    • Substrate <--------> Product
    • Enzymes increase rate of forward and reverse reactions
    • do NOT change equilibrium
  52. Energy of Reactions (w/ and w/o enzymes)
    • NO enzyme: S <---> P (SLOW)
    • with enzyme: S<---> ES---->P (ES complex intermediate)
    • All reactions- substrate must be activated
    • Enzyme catalysis- lowers the amount of energy needed for activation, forms ES complex
  53. Reaction profiles
    (SEE NOTES!!!)
  54. Enzymes work by:
    • Enzymes increase reaction rates by lowering activation energy
    • stabilize transition state
    • specifically bind substrate
  55. Carbonic anhydrase in RBC
    • fastest enzyme
    • increases reaction rate by factor of 10million
    • CO2 + H20 <---CA--> HCO3- + H+
  56. How enzymes increase reaction rate
    • substrates in close proximity
    • in correct orientation
    • transition state stabilized
    • creates a focal point
  57. Enzyme specificity
    • Substrate binds at active site generated by 3D folding
    • Weak interactions with R groups
    • 3D folding and amino acids residues important to enzyme specificity
    • Enzyme may bind 1 or more substrates
    • ex: Hexokinase (low specificity- C6 sugars), glucokinase (very specific- only binds glucose)
    • different binding sites
  58. 1y sequence and structure (enzymes)
    • folding brings active site residues together
    • may be far apart in 1y sequence (eg cysteine proteases)
    • similar active site sequence and folding- similar activity
  59. Reaction conditions
    • pH- affects ionisation state of amino acid R groups, affects interaction between E+S
    • Temperature affects 3D structure
  60. Nomenclature
    EC numbers- enzyme classification
  61. Isoenzymes
    • catalyse same reaction
    • different 1y sequence or subunit composition
    • eg: lactate dehydrogenase- different subunit compositions in different tissues (Muscle = M type, Heart = H type)
  62. Coenzymes
    • HELP enzymes
    • play essential part in reaction
    • Derived from vitamins (NAD+ from niacin- Vit B3, FMN and FAD+ from riboflavin- Vit B2)
    • (glycolysis)- G3P dehydrogenase
    • Gly-3P <------> 1,3 BPGlycerate
    • NAD+ ^ NADH
    • NAD+ is a coenzyme- not catalyzing but there to accept a H
  63. Enzyme Cofactors
    • Usually metal ions
    • eg: Ca2+, Fe2+, Cu2+, Mn2+
    • help in bonding of substrate in active site
    • EDTA- metal chelator- enzyme inhibitor (can be used to inhibit blood clotting)
  64. Hyperbolic curve (enzyme reactions)
    • At constant concentration of enzyme
    • reaction rate increases with increasing [S] until max rate is reached
    • uncatalysed reactions do NOT show this saturation effect
    • saturation occurs due to all enzyme sites being filled with S ------> ES complex
  65. Michaelis-Menten Kinetics (Km)
    • Michaelis constant
    • S concentration when rate is 1/2 Vmax
    • units: M, mM, uM
    • Low [S], rate linearily proportional to [S]
    • high [S], rate independent of [S]
  66. Km
    • Low Km- low [S] when 1/2 Vmax indicates enzyme with high affinity for S
    • High Km- High [S] when 1/2 Vmax indicates enzyme with low affinity for S
    • (Km is dissociation constant)
  67. Isoenzymes (Km)
    • Can vary in Km (affinity for substrate and specificity)
    • Hexokinase- Km LOW- high affinity
    • Glucokinase- Km HIGH- Low affinity
  68. Biological importance of Km
    • Hexokinase (low Km)- in many cell types efficient in hexose metab.- not specific to glucose, Glu metabolised at low blood glucose levels (impt in energy in brain)
    • Glucokinase (high Km)- in liver (only), glu metabolized only when blood glucose levels high (glucose storage as glycogen
  69. MM Equation
    • V=Vmax [S]
    • [S]+Km

    • [S]=Km, V=Vmax
    • 2
  70. Lineweaver-Burk Plot
    • Conversion of Michaelis-Menton Kinetics to straight Line
    • calculate activity for known [S]
    • 1 = 1 + Km 1
    • V Vmax Vmax [S] (y=C=mx)
    • Intercept= 1/Vmax
    • slope= Km/Vmax
  71. Measuring Enzyme Activity
    • E+S <-------> ES <------> E+P
    • measure initial reaction rate
    • usually spectrophotometrically
    • (substrate loss OR product formation)
    • Standardise with known amount of pure enzyme
    • IU= activity required 1 umol S --> P per min
  72. Measuring Activity (enzyme)
    • Reaction conditions- keep standard
    • High [S], reaction not limited
    • High coenzyme concentration
    • constant pH (buffer)
    • Constant temp
  73. Use of Enzyme inhibitors
    • Drugs- control inappropriate enzyme activity
    • eg: inhibitors of blood clotting enzymes, protease inhibitors to control HIV replication, penicillin to prevent bacterial cell wall growth
    • Drug design-more effective/specific drugs (3D modelling important)
  74. Enzyme inhibitors (Types)
    • Reversible
    • irreversible
  75. Irreversible enzyme inhibitors
    • eg: organophosphates binding to acetylcholinesterase
    • AchE-removal of Ach neurotransmitter
    • inhibition- constant neuromuscular stimulation (resultin in paralysis)
  76. Reversible Enzyme inhibitors
    • (competitive and non-competitive)
    • rapid dissociation from enzyme
  77. Competitive Reversible enzyme inhibitors
    • Bind AT active site
    • compete with substrate for binding
    • S and I often similar structures
    • Less ES comples- reaction rates DECREASES
    • CAN reach Vmax, more S needed
    • Km is INCREASED (rate and equation affected by I)
    • Vmax is same
    • Km higher (becuase you need more S)
    • Product can be competitive inhibitor (feedback inhibition-regulation)
    • Eg competition tx for poison ingestion
  78. Non-competitive Reversible Enzyme inhibitors
    • DOESN'T bind at active site
    • DOESN'T compete with S binding
    • E+S <---> ES
    • E+I <---> EI
    • E+S+I <---> ESI
    • Less product formed in presence
    • CANNOT reach Vmax even with hight [S]
    • CANNOT overcome inhibition by adding more substrate
    • Vmax is lower
    • Km is same
  79. Allosteric Enzymes (sigmoidal curve)
    • More than 1 substrate binding site
    • change shape when substrate binds
    • co-operative binding- 'other shape'
    • 2 subunit enzyme
    • T form (tense)- low affinity
    • R form (relaxed)- high affinity
    • S binding to one subunit increases affinity for 2nd site
    • bind to subunits away from active site alter affinity for substrate
  80. Allosteric activators vs allosteric inhibitors
    • Activators: bind to R form, increase rate S binding to E
    • Inhibitors: bind to T form, decrease rate S binding to E
    • Enzyme activity can be altered- respond to physiological changes
Card Set
Proteins and Enzymes