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University of Cambridge
MVST Part IA Molecules in Medical Sciences
Biological macromolecules, protein structure and enzyme catalysis
Dr Helen Mott

Lecture 1:
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Glucose = aldose at C1, 4 chiral centres (C2, C3, C4, C5), Beta chair=2 conformations

•

Monosaccharides more than 5C usually cyclic > new chiral centre > α- and β-ring
enantiomers
o fructose = ketose at C2

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Free end of chain = reducing end > ring can be opened to produce free reducing aldehyde
group

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Glycogen more ends to cut glucose – accessible – short term store – water between units

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Cellulose – beta 1,4 alternate aspect > very straight chained > held by H-bonds to form
microfibrils

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Chitin = N-acetyl glucosamine
...
4nm/10 nucleotides

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tRNA: single-stranded but some areas complementary > hairpin loops > 4 arms

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AA: alpha carbon > L-form

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Hydrophobic aliphatic: cluster away from water, pack tightly: ALVPIG
o Proline: rigid ring > bends and kinks
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Lecture 2: Protein 3D structure
Primary structure (1°) = linear sequence of AA > determines overall structure
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Experiments: Sample RNAase was unfolded/denatured in test tube by adding urea
(disrupts the non-covalent forces) and mercaptoethanol (reduces disulphide bonds) >
denaturing agents removed > spontaneously refolded > active = native structure had reformed
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Lecture 3: Protein structure vs function
➢ Co-factors: reactivity not in AA side chains, from vitamin and minerals, carrying e- or O2, coenzymes
➢ Prosthetic group: tightly bound, required for structure e
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haem, FAD (riboflavin)
• Oxygen binds to central Fe2+
• Carry e- in Fe2+/Fe3+
➢ Co-substrate: used once, released and regenerated e
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NAD+ (niacin), Co-A
➢ Hb, Mb: hydrophobic pocket for correct geometrical haem binding
• Mb: 153 AA, 8 a-helices > hyperbolic saturation > diffusion within muscle tissues
➢ Hb: tetramer with 2 alpha and 2 beta subunits
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VH and VL form antigen binding site
• Extra loops=complementary determining regions (CDRs), rest B-sheet
• CDRs determine specificity, 3 on each chain > recombination
• Light chain has one constant domain = CL binds to CH1
• CH2, CH3(Fc) = dimerization + interact with receptors, different effector functions

Lecture 4: Enzyme Reaction Rates
➢ Enhance by more than billion-fold
➢ Operate at body temp (37°), 1atm, neutral pH (depending on their function) unlike industrial
catalysts
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➢ Instead it reduces the energy of the transition state (TS), reducing the activation energy and
overcoming the kinetic barrier
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Free energy
needed to overcome this is provided by the binding energy
• when substrate binds to active site, many VDW forces > max binding energy released
when TS formed
4) Substrate strain = push substrate towards TS when bound, enzyme structure more
complementary to TS than substrate
•
•
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These occur at active site where substrate binds selectively – AA chiral so can select
chiral compounds
products are released rapidly (EPC not too stable)
Induced fit: enzyme and the substrate both change conformation when they
interact
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2 kJ/mol stabilisation at 25° = 2
H-bonds
Carbonic anhydrase
➢ X-ray diffraction studies indicate Zn2+ bottom of 15-Å deep cleft
• Co-factor
• Lowers pKa of bound water from 15
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g
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They are far apart in primary structure but come together in tertiary structure
• Ser195 with OH

•
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His57 acid-base catalysis, pKa closes to physiological pH, readily loses and accepts
proton
Asp102 negatively charged, hydrogen bonding

1
...
Step 2: General acid catalysis aids breakdown of tetrahedral intermediate to acyl-enzyme
intermediate
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Step 3: C-terminal NH2 leaves (weak affinity, changed structure) and replaced by water form
solvent (abundant)

4
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➢ In effect, the oxyanion hole is only filled when the transition state is formed
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• When substrate binds to enzyme, carbonyl C constrained from binding in oxyanion
hole
• In tetrahedral intermediate the carbonyl oxygen is charged so enters hole and Hbonds to NH groups of backbone on Gly193 and Ser195
• Conform change allows aa before the peptide bond to form unsatisfied H-bond to
Gly193
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➢ Next to the active site there is a selectivity pocket that selects the correct substrate
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Aspartate proteases:
➢ HIV protease
➢ These use aspartic acid side chains as the reactive groups
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➢ Their pKa is usually 3-4 i
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they readily lose their proton at neutral pH, at zwitterion
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2 Asp residues in active site – Asp 1’s environment favours
protonation, Asp2 is in aqueous and deprotonated
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g
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Lecture 5 Summary: Enzyme kinetics and control
➢ Oxidoreductase: redox with movement of e➢ Transferase: functional group: methyl, acetyl, phosphate e
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kinase
➢ Hydrolase: break covalent bonds with water, e
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g
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g
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g
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Only binds substrate OR inhibitor
• Decreases [active enzyme], therefore need increased [S]
• Increases Km, half Vmax needs higher [S] without altering Vmax
• Use Lineweaver-Burk plot to confidently extrapolate hyperbolic function to find
Vmax at non-physiological [S]
➢ Non-competitive: binds to E and ES, reversible, alters important catalytic residue but not
those that bind to substrate
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g
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AS between two domains
• Glucose sensor in B-cells of pancreas > insulin release
• Mutations cause neonatal diabetes and MODY
• Allosteric activator stabilises active conform of enzyme

➢ Allosteric regulators bind at separate site, change shape of active site
• Homotropic: binding substrate to AS change conform of enzyme to high activity,
cooperative change leads to sigmoidal kinetics
...
Each monomer has AS, allosteric sites at subunit interface
• F6P binds cooperativity stabilising high affinity state, sigmoidal
• ATP preferentially binds to allosteric site in T low affinity conform
• AMP reverses inhibition
➢ Phosphorylation can also change conform > activity
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g
...
g
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g
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Isothermal titration calorimetry > ∆H when titrating ligand into protein solution
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Surface plasmon resonance > ligand binding changes the refractive index of light

➢ Molecular recognition changed by altering shape (more complementary) /chemical
properties of binding surface (phosphorylation)
➢ Growth factor signalling: RTK > SH2 binds to phosphotyrosine > Gp > kinase cascade >
transcription
➢ Epidermal GF receptor – ligand induced dimerization
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Cell survival, growth, proliferation, differentiation, inductive signal in development

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Domain 2 has beta hairpin that interacts with domain 4

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Ligand binding rotates 1 and 3; join to form EGF binding site

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New surface exposed on 2 – beta hairpin interacts with domain 2 on another
monomer

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Asymmetric, only 1 kinase active, phosphorylates both tails

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Grb2 (adapter protein that couples domains) has SH2 that binds to activated
receptor
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❖ Raf doesn’t detect GTP
❖ Ras displaces N-terminal region to activate kinase phosphorylates Ser+Thr SC



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