Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Amino Acids
30 September 2015

11:40

History
• First amino acid isolated was asparagine in 1806
○ Louis-Nicolas Vauqelin
○ Extracted and isolated from asparagus
• Term amino acid introduced in 1898
• More than 200 discovered, only 20 found in protein

Alpha, Beta, Gamma - amino acids
• Carbons extending from the carboxylic carbon are labelled with greek letters
• Amine group can be attached to alpha, beta, or gamma
○ Alpha amino acids are found in proteins

•

Weak acids and bases

•
•
•
•
•

pKa = -log10(Ka)
Alpha-carboxylic range: 2-2
...
86, glu=4
...
6
Higher pKa, lower pH

Effect of pH on Amino Acids
• Le chatelier's principle
○ Alter equilibrium and the system will try to correct the change

•

• 1
...
equilibrium moves right
• Zwitterion
○ Molecule with 2 charges groups
○ Low pH, no charge on COOH (1)

Proteins ^M Enzymes Page 1

○ Low pH, no charge on COOH (1)
○ High pH, no charge on NH3 (2)
○ Intermediate both charged

○

pH and Amino Acids
• pH affects polarity of zwitterions
• Side chain pKa affects folding and structure

Stereoisomers

•

•

•
•
•
•

Molecules with a chiral centre (C with 4 different groups)
Different plane polarised light diffraction
L (laevorotatory)-amino acids are most proteinogenic
D (dextrorotatory)-amino acids found in sea dwelling organisms and bacterial cell wall

•

Electronegativity
• Tendency of an atom to attract electron bonding pair
○ Shown on the Pauling Scale

•

• Intermolecular Forces
○ Polar
 Permanent dipoles
 Hydrogen bonding (hydrophilic)



○ Covalent
○ Van der Waals
 Temporary dipoles - weak interaction
 Hydrophobic



Proteins ^M Enzymes Page 2

20 Amino Acids (+2)

•

•

•

•

•

Proteins ^M Enzymes Page 3

•

•

•

Essential Amino Acids
• Isoleucine
• Leucine
• Lysine
• Methionine
• Phenylalanine
• Threonine
• Tryptophan
• Valine
• Lysine and methionine can be limiting

Detection of Amino Acids
• Spectrophotometric assay
○ React with ninhydrin
○ Ruhemann's purple produced - can be measured with colorimetry
• Aromatic amino acids absorb UV260-280nm
• Mass spectroscopy
Proteinogenic and Non-proteinogenic
• Proteinogenic
○ ‘Protein building’ – amino acids within proteins
○ standard amino acids– 20 which are directly encoded for by codons (3 bases) in the
genetic code
○ Two non-standard- proteinogenic amino-acids, directly encoded- selenocysteine,
pyrrolysine
○ ‘Protein building’ – amino acids within proteins

• Non-proteinogenic
○ Not found in proteins
○ Not directly encoded by genetic code

Peptides
• Linear polymers of amino acids
• Properties determined by amino acid sequence
• Written from NH to CO
• Synthesis
○ Condensation (amide bond)

Proteins ^M Enzymes Page 4

○

○

○ Reversible, but stable (reverse reaction very slow)

Proteins ^M Enzymes Page 5

Peptides
06 October 2015

10:05

• Linear polymers of amino acids
○ Properties determined by amino acid sequence
• Amino acids termed residues
• <50 amino acids = oligopeptide
• Named from the N terminal residue
• 50-2000 residues
• Mean Mr of amino acids is 110Da
• Proteins range from 5kDa to 250kDa
Peptide Synthesis
• Condensation
• Peptide bond

•

SP2 and SP3 bonding
• SP3 - 109o bond angles, sigma bonds

•

• Sigma C-H bonds, sigma C=C bond
• P orbitals of C atoms interact to create pi bond
• This bond is rigid and cannot rotate
Cis and Trans Isomerism

Proteins ^M Enzymes Page 6

• Structural Features
○ Resonance

 C-N 40% double bond character
 Resonance between C=O and C-N
□ More rigid
□ No free rotation
□ Isomerism
 Trans isomers favoured
○ Proline
 Imino acid
 Only trans synthesised
□ Prolyl isomerase can make cis form



Ramachandran Plot
•
A Ramachandran plot (also known as a Ramachandran diagram or a [φ,ψ] plot), originally
developed in 1963 by G
...
Ramachandran, C
...
Sasisekharan,[1] is a way to
visualize backbonedihedral angles ψ against φ of amino acid residues in protein structure
...
wikipedia
...
Adjacent amino acids can adopt different configurations by
rotation around the two other bonds in the backbone
...
These angles are measured in degrees where 180° is the angle of the bonds when all
of the atoms of both residues lie in the most extended conformation
...

Proteins ^M Enzymes Page 8

is positive so the values go from 0° to 180° and in the other direction they go from 0° to -180°
...
)
From ...
co
...
html>

Most of the amino acid residues in a given protein are found in some form of secondary
structure such as α helix,β strands, or turns
...
This is why the structure is so regular
...
Since the residues in a β stand are in an extended form, the Φ
and Ψ angles in this conformation are close to 180°
...
These can
be plotted on a diagram called a Ramachandran plot, named after the biophysicist G
...

Ramachandran (1922 - 2001)
...

Similarly, residues in a right-handed α helix have very similar bond angles around Ψ=-45°, Φ=+
45°
...
Some regions of the
Ramachandran plot will be empty because of steric clashes between the oxygen atoms
[see The Peptide Bond]
...

From ...
co
...
html>

Alpha-helix and Beta-sheet
• 1951 7 papers
• 2o structures proposed
• x-ray crystallography
• Linus Pauling - 1954 Nobel Prize
• Beta-sheet
○ Parallel sheet
○ h-bonds to 2 different amino acids

○

○ 2 or more polypeptide chains - beta-strands
○ Extended
○ Side chains point opposite directions
2-10 chains (4-5 normal)
Proteins ^M Enzymes Page 9

○ 2-10 chains (4-5 normal)
• Alpha-helix
○ Pauling and Corey predicted
○ Fully exploited H bonding of CO-NH
○ Every 4th amino acid is a full loop
 Each residue rotate 100o

○

○
○
○
○

Each residue rises by 1
...
Only 1400 recognised - limited in
nature
• Common motifs and folds present in many proteins (even unrelated amino acid sequence)
• Domains: recognisable fold with known function

Proteins ^M Enzymes Page 12

Tertiary Structure and Protein Classes
07 October 2015

11:49

Alpha-keratin
• Alpha-helical proteins which entwine into coiled coils through R group interaction
• Protofilament
• Soft or hard depending on cysteine content (disulphide bridges)
○ Soft (skin) low cysteine content
○ Hard (horns) high cysteine content
○ Human hair ~14% cysteine
Porins
• Large β-barrel composed of 16 antiparallel sheets in barrel shape

•

• Channels through membranes - non-polar environments
• Hydrophobic groups to outside
• Hydrophilic groups inside - filled with water
Myoglobin
• Globin fold
• Similar structure to haemoglobin
• 1st protein structurally resolved
○ X-ray crystallography 1958
• 153 residues
• Compact molecule
• 8 alpha helices connected by loops
• Metallic prosthetic group
• Water soluble
• Non-polar residues internal
○ Leucine, methionine, phenylalanine
• 2 internal polar residues - histidine, covalent bond with Fe2+
Haemoglobin
• Oxygen transport metalloprotein
• Vertebrates and some invertebrates
• 97%DM of blood cells
• 1
...
g
...
g
...
g
...
Reacts with phenolic residues
○ Bradford assay - coomassie blue dye
• Isoelectric Focussing
○ Charged protein migrates down gel to point of neutral charge
 Balance between dissociation of acid and basic groups
○ Every protein has a different number of acid/basic groups
 PI point is balance

○

Proteins ^M Enzymes Page 17

Protein Sequence
• Chemical or enzymatic
• Must be relatively small in size
• Separated before further analysis
• Most methods relatively specific
• Cyanogen bromide:
○ Chemical
○ Carboxyl side of methionine residues cleaved
• Trypsin
○ Enzymatic
○ Carboxyl side lys and arg
○ Proteins containing 9 lysines and 7 arginines will give 17 peptides

Separation by chromatography
• Amino acids/short peptides separated by chromatography
• AAs/peptides elute at different times
• Measured with detector
Protein Sequencing
• Frederick Sanger sequenced Insulin in 1952
○ Insulin widely available (at Boots)
○ Took 10 year stop sequence - separate subunits
○ Breakthroughs
 Cleaved into separate AAs
 Labelled N-terminal amino acids
○ Demonstrated 2 n-terminal AAs - 1 glycine and 1 phenylalanine
○ 12k molecular weight
• Pehr Edman developed sequencing technique - 1950
○ Current techniques still based on his
○ Automated in 1967
○ Label N-terminal aa of peptide, cleave and identify
○ Different segment from different cleavage enzyme/treatment

○

• Protein Sequence Alignment
○ Trypsin cleaves c-end of lys and arg
○ CNBr cleaves c-end of met
○ Different peptide segments are generated
Overlapping short peptides
Proteins ^M Enzymes Page 18

○ Overlapping short peptides
○ Can be aligned to give larger sequence
• Location of disulphide bridges
○ Method by Brown and Hartley
○ Tryptic digestion of protein and separated along 1st dimension
 S-S intact
○ Peptides treated with performic acid and separated by electrophoresis, repeated along
2nd deimension (breaks S-S)
○ Peptides not S-S linked will migrate the same distance
○ Peptides cleaved will give 2 new spots
 These are purified and S-S location is established

○

Protein Configuration in Space
• 1-100nm - hard to visualise in 3D
○ 400-700nm visible
○ 200nm with oil lens max resolution
• x-ray range 0
...
1-100Angstroms)
○ Average atomic distance 1
...

 Gives split signal
 Rule = number of H on neighbouring C plus 1

Proteins ^M Enzymes Page 20

Rule = number of H on neighbouring C plus 1





○ NMR of proteins
 Aqueous samples of high concentration pure protein
 Recombinant proteins easier to produce and can have active isotope labels added
 Sample in buffered solution in thin walled glass tube
 Measurement time varies
 Most protons resolved in proton by 1D (deuterium) 1H NMR
 H in water gives strong signal, so deuterated water must be used 2H2O (D2O)
 We can study how bonds shift with pH etc
...
31JM-1K-1
• Free energy and equilibrium constant
○ At equilibrium ΔG = 0
0 = ΔG0' + RTln

R = 0
...
303RTlog10K'eq
Proteins ^M Enzymes Page 24

ΔG0' = -2
...
303RT = 10-ΔG0'/5
...
69kJ/mol
○ DHAP --> GAP during glycolysis
 At equilibrium K'eq = 0
...
303RTlog10K'eq
= -2
...
008315x298xlog10(0
...
53kJ/mol
Endergonic - not spontaneous
 When [DHAP] = 2x10-4 moles and [GAP] = 3x10-6 moles
ΔG = ΔG0' + RTln
= 7
...
303RTlog10
=7
...
41kJ/mol
= -2
...
g
...
01-1
...

○ Units of k = s-1
• If 2 substrates then 2nd order
○ A+B --> P
○ V = k[A][B]
○ K = M-1s-1
○ Sometime appears to be 1st order
 One S may be saturating, so doesn't appear to affect the rate
• At high [S] a reaction may appear independent of [S] because E is saturated - 0 order
Multiple Substrates
• Most biochemical systems start with 2 substrates and yield 2 products
• Can be classified sequential or double displacement
• Sequential
○ S bind with E to form ternary complex before product released
○ Binding ordered or random
○ Many enzymes binding NADH exhibit ordered sequential

○

• Double-displacement
○ 1 or more products released before all substrates bind
○ Defining feature is formation of a substituted enzyme intermediate in which the enzyme is temporarily
modified

○

Allosteric Enzymes
• Don't follow M-M rules
• Consist of multiple subunits and active sites
○ Binding of substrate influences active site for other substrate to bind
• Show sigmoidal not hyperbolic V0/[S] plot

○

Proteins ^M Enzymes Page 30

Enzyme Inhibition
21 October 2015

12:10

• Small molecules/ion bind with enzymes
○ Inhibitory
○ Enzyme-substrate complex
• Variety of uses
○ Biological control
○ Medicinal control
○ Toxic attack
• Vital info about mechanisms
• 2 classes
○ Irreversible
○ Reversible
Irreversible Inhibitor
• Dissociates very slowly from target
○ Bind tightly
• Bound covalently or non-covalently
• Drugs --> irreversible inhibitors
○ Penicillin
○ Transpeptidase (bacterial cell walls)
○ Aspirin
○ Cyclooxygenase - inflammation
• Cysteine residues vulnerable
Reversible
• Rapid dissociation
• Weaker binding
• Useful in design of drugs
○ Basis of 1st antibiotic
○ Sulphonamides and dihydropteroate synthase (DHPS)
• 3 main types
○ Competitive
 EI complex formed
 Inhibitor resembles the substrate
□ Binds to active site
 Catalysis diminished
 Can be relieved by greater [S] - increased apparent Km
 Cancer drugs
□ Methotrexate resembles dihydrofolate reductase (nucleotide replication)
 Ki (inhibition constant) = [I][E]/[EI]
□ Smaller ki = more potent inhibition
 Vmax not changed - just need more substrate
□ Apparent Km (Kmapp) increased

Proteins ^M Enzymes Page 31





○ Uncompetitive
 Anti-competitive inhibition
 I binds to ES only
 Slows catalysis
□ Slower P release
 Forms ESI complex
 No product
 Lowered Vmaxapp
 Increasing [S] has no effect
 Few examples in single substrates



○ Noncompetitive
 I and S bind to E
Proteins ^M Enzymes Page 32

 I and S bind to E
□ Different binding sites
□ Simultaneous binding - no overlap
 Proteins flexible
□ Alter shape of molecule
 Reduces catalysis by reducing Kcat (molecules/sec)
□ Not formation of ES complexes
□ Proportion of E binding to S unchanged
 Increased [S] has no effect
 Lowered apparent Vmax
 Km unchanged
 Lowers functional [E]
□ dilution





○ Mixed Inhibition
 Effects of inhibitor don't fall into the 3 categories
 Inhibitor acts as
□ Non- or uncompetitive
□ Competitive inhibitor
 Reduces
□ Binding of E and S
□ Kcat

Irreversible Inhibitors - Mapping the Active Site
• Determine catalytic groups
• Irreversible inhibitors can modify catalytic groups
• 3 groups
○ Group-specific
 Covalently binds to a group, results in changed conformation

Proteins ^M Enzymes Page 33



○ Reactive substrate analogues
 Resembles S
 More reactive chemically
□ Binds to nearby amino acids so we can see



○ Suicide inhibitors
 Normally resembles S
 Binds in active site
 Locks E into dead-end product
□ Covalent modification
 Many drugs
 Monoamine oxidase (neurotransmitter) inhibited by N,N-dimethyl propargylamine
○ Transition State Analogues
 Substance mimics transition state
□ Potent inhibitors (Pauling 1948)
□ Geometry of transition state
Greater affinity for E

□ Lactone analogue of tetra-NAG
 Isomerisation of L-proline to D-Proline
□ Proline racemase
□ Pyrrole 2-carboxylate transition state analogue

□

Proteins ^M Enzymes Page 34

Proteins ^M Enzymes Page 35

Catalytic Strategies - Covalent and Acid/Base
27 October 2015

10:05

• Substrate bound
○ Catalytic functional groups
○ 4 strategies
• Covalent catalysis
○ Active site binds covalently with substrate
○ Nucleophile (electron pair donor)
• General acid base catalysis
○ Proton donating or accepting
○ Not H2O as a donor
• Catalysis by approximation
○ Bringing reactive substrates together
• Electrostatic/metal-ion catalysis
○ Redox reactions and/or increased binding energy

Proteases
• Cleave proteins - hydrolysis
○ Addition of water molecule to peptide bond
○
• Hydrolysis of peptide bonds is thermodynamically spontaneous
○ Reaction very slow - 10-100s of years without catalyst
○ Milliseconds in presence of an enzyme
• Nucleophilic attack of the carbonyl group
• Families of proteases
○ Single amino acid substitution studies
 Importance of certain amino acids in catalytic group
○ 4 major functional groups of proteases
 Defined in relation to active moieties
Serine

Covalent and acid/base

Cysteine

Covalent and acid/base

Aspartate

Covalent and acid/base

Metalloproteases Electrostatic and metal-ion
○ Serine proteases - Chymotrypsin
 Breakdown of protein in the digestive tract
 Cleaves peptide bonds selectively
□ Carboxyl terminal side
□ Large hydrophobic amino acids
 Covalent/acid-base catalysis
 Nucleophilic serine attacks unreactive carbonyl carbon of substrate
□ Binds covalently
 Chromogenic substrate (easy to monitor colour change)
□ Kinetics - evidence to catalytic serine
□ N-acetyl-L-phenylalanine p-nitrophenyl ester
 Hydrolysed to yellow p-nitrophenolate
□ Under steady state conditions obeys M-M kinetics
 Km 20µM Kcat 77s-1
□ Stopped flow method
 Initial burst of colour
 Followed by slower release rate to eqm
□ Indicates 2 step reaction
 Step 1 - nucleophile in enzyme attacks peptide bond; split off c-terminal half, N-terminal half
bonded to enzyme
...


◊

 Decreased overall ΔG
 Enzyme-protein attachment
□ Chymotrypsin-substrate peptide
 Groove on surface
Proteins ^M Enzymes Page 36

 Decreased overall ΔG
 Enzyme-protein attachment
□ Chymotrypsin-substrate peptide
 Groove on surface
□ Weak H bond forms between groove amino acids and substrate
□ Strong binding of target residue
 Adjacent side chain fits into S1 hydrophobic pocket, lining target residue up with active site
 Specificity depends on amino acid directly N-terminal side of the bond
□ S1 pocket explains specificity
 Chymotrypsin's catalytic group
□ Catalytic triad
 3 residues



□ Aspartate 102
 -ve charge
 Isolated from exterior
□ Histidine 57
 +ve charge
 Weak base
□ Serine 195
 Hydroxy side chain - nucleophilic
 Required to lose a proton (his57)
□ H-bonding
 Serine side chain to his imidazole ring
 Imidazole ring to carboxylate of asp
□ His57 orientate ser195
 Polarise hydroxyl group
 Alkoxide ion
□ Asp102 orientate his57
 Improved proton acceptor
 Chymotrypsin mechanism step 1

□

□ Fast burst
□ Cooperative action of the triad
 First transition state
□ His57 acts as a base
 Removes a proton from ser195
□ Nucleophilic ser195 attacks the C=O of the substrate
□ -ve Asp102 stabilises the +ve charge of His57
 Oxyanion hole (stabilises negative charge on substrate created by tetrahedral binding of C)
◊ Strong H bonds
◊ Helps reach transition state 1



□ Transition state breaks up
 His57 acts as acid
◊ Donates proton
◊ N-H group on substrate
 Peptide bond
 C-terminal half free to leave
 N-terminal bound to Ser195

Proteins ^M Enzymes Page 37

 N-terminal bound to Ser195





 Chymotrypsin mechanism step 2

□

□ Water enters active site
 c-terminal removed
 Hydrolysis of ester
□ His57 acts as base
 Steals proton from water
 Produces nucleophilic OH◊ Attacks carbonyl
 His general acid catalyst
□ Unstable tetrahedral transition state
 Breaks up - HisH+57 acting as acid
 Donates proton back to ser195



□ Breaking Serine-substrate bond
□ N-terminal half of S carries target aa as new c-terminal
□ Formation of carboxylic group
 Moves C=O
□ Enzyme recharged

Proteins ^M Enzymes Page 38

□ Enzyme recharged



 Importance of pH
□ Effective His57
 pKa of 6
...
1mM
 Enhances specificity and binding energy (-ve charge)
○ Approximation excludes water
 Successful transfer requires no H2O
□ Phosphorylates more easily than NMPs
 Induced fit binding
□ P-loop closes - structural change
□ Fold over and holds phosphate
○ Other uses of approximation
 Enzyme serves as template
□ Bind substrates
□ Close proximity in reaction centre
 NMP kinase
□ NMP and NTP
□ Stabilises transition state
 Bring substrate into contact with catalytic group or other substrate
□ Increased encounter rate
 Freeze translational and rotational motion

Proteins ^M Enzymes Page 42

Enzyme Regulation
03 November 2015

10:08

• Enzyme activity
○ Regulated
○ Ensures function at time and place
• Regulation essential
○ Coordination of biochemical processes
• Taking place all the time and in all organisms
5 strategies
1
...
Isoenzymes
○ Multiple forms - different Km and Vmax
3
...
Zymogen
○ Proteolytic cleaving - inactive form
5
...
Allosteric Control
• Control by
○ Binding an effector molecule
○ Allosteric site
• Distinctions
○ Regulatory/allosteric site
○ Catalytic/active site
• Allosteric and catalytic subunits (4o structure)
○ Separate polypeptide chains
• Allosteric effectors
○ Positive effect - Activators
 Enhances attraction
□ Substrate or other catalytic site
 Not limited to enzymes
□ O2 and haemoglobin
□ Substrate and effector
 Binding to one subunit
□ Conformational change
□ Interacts with remaining active sites
□ Enhance O2 affinity
□ Cooperation efficiency
□ Hb allosteric activation
 4o structure consists of 2 alpha, 2 beta
 Binds O2 - left Fe atom, electron rearrangement, fits closer into molecule, tugs His
 His protein chain - alpha helix, 15o rotation
 Binding O2 - alters interface, additional O2 binding (20 fold increase)
○ Negative affect - Inhibitors
 Binding of effector reduces affinity
 O2 affinity in pure Hb much higher than in blood - O2 released more from blood into cells
 2,3-bisphopshoglycerate
□ Binds to allosteric site on Hb
□ Reduces affinity to O2
 Feedback mechanisms
Proteins ^M Enzymes Page 43

 Feedback mechanisms
□ Preferentially to deoxyhaemoglobin
□ Structurally unmodified
 ATCase - feedback control
□ Aspartate transcarbamoylase
 2 regulatory chains
 3 catalytic chains
□ Catalyses 1st step in pyrimidine synthesis

□

□
□
□
□
□

Condensation reaction
Committed step is the ultimate formation of nucleotides
ATCase inhibited by CTP (cytidine trisphosphatase) - end product
CTP structurally different to active site - not competitive inhibitor
PALA analogue used to test effects (competitive inhibitor
 2 4o states (with and without PALA) - mechanism of regulation
□ 2 models
 Enzyme either tense or relaxed
◊ Relaxed binds S more readily, T has lower affinity
◊ T much more compact, relatively inactive - equilibrium state of ATCase favours T
(factor of 200)
◊ PALA causes shift to R
◊ CTP favours T, carbamoyl phosphate and aspartate favour R
 Concerted Model
◊ Enzyme subunits all connected
 Conformational changes conferred to all
◊ All R or all T
 Sequential
◊ Subunits less connected
 Varying states
◊ Effector
 Induced fit to sequential changes
 Influences other adjacent subunits
 Change state and increases affinity
 Combination of models generally the case in practice
2
...
Reversible Covalent Modification
• Covalent attachement
○ Another moelcule modifies enzume activity
• Donor molecule
○ Functional moiety
○ Enzyme properties
• Most common types
○ Phosphorylation
○ Acetylation
• Regulation of acetyltransferase and deacetylase
○ Regulated by phosphorylation
• Most are reversible
• Phosphorylation
○ Most common regulatory mechanism
 Intracellular proteins
 30% eukryotic protons phosphorylated
 Reversible
Enzymes/membrane channels
Proteins ^M Enzymes Page 45

○ Enzymes/membrane channels
○ Catalyse reactions
 500 kinases in humans
 Kinome
 Multiplicity - fine tuning metabolism
○ Transfers terminal g phosphate group
 Ser and Thr
 Tyr
○ Phosphatase
 Catalytic removal of phosphate group
 Trn off signalling pathways (activated by kinases)
○ Phosphorylation and dephosphorylation
 Essentially irreversible reactions under physiological condition
 Without enzymes = negligible
○ Phosphorylation only when
 Specific kinase
 ATP cleavage
○ Net process of 2 reactions
 Hydrolysis of ATP
 ADP + Pi
 Highly favourable (free energy) - unidirectional

• Phosphorylation is a highly effective means of control
○ Protein activation - structural/thermodynamic/kinetic/regulatory
○ Negative charge
 Addition to protein - new electrostatic interactions
 After substrate binding/catalytic activity
○ Hydrogen bonds
 Phosphoryl group increases H bond formation
 Tetrahedral geometry
○ Free energy
 Increase free energy of enzyme
 Changing conformational state
○ Rapid - less than 1 second, but can take hours if needed
○ Highly amplified effects
 Reaction amplified
 Single t hundreds
 Activation of enzymes
○ ATP cellular currency
 Links energy states
 Enzyme activity
 Regulation of metabolism
• Protein kinase specificity
○ Dedicated protein kinases - single protein
○ Multifunctional protein kinases
 Protein kinase A
 Many proteins
 Coordination
Proteins ^M Enzymes Page 46

 Coordination
○ Determination of specificity
 Sequence surrounding Ser or Thr
 Consensus
□ Arg-Arg-X-(Ser/Thr)-Z
□ Variations
□ Distant residues
 Activation of protein kinase A
□ PKA - covalent/allosteric
□ Fright, fight, flight reaction
 Adrenaline
 Adrenaline triggers cAMP formation
◊ Intracellular messanger
 cAMP activates PKA
◊ Altering of 4o structure
◊ Phosporylation
□ PKA is a holoenzyme - subunits R (regulatory) and C (catalytic)
 Without cAMP R2C2 structure
 cAMP causes dissociation to R2 and 2xC
 R chain pseudosubstrate - blocking C active site



4
...
l
• Digestive enzymes
○ Pepsinogen --> pepsin
○ Trypsinogen --> trypin
○ Proelastase --> elastase
• Zymogen systems
○ Blood clotting
 Cascade of zymogen conversions
 Rapid response
○ Hormones
 Transcribed as zymogens
 Proinsulin - activated by cleavage
○ Collagen
 Major bodily constituent
 Transcribed as procollagen
○ Developmental stages
 Metamorphosis
 Parturition (uterus post-birth)
Proteins ^M Enzymes Page 47

 Parturition (uterus post-birth)
 Rapid breakdown of collage
 Procollagenase
○ Apoptosis
 Programmed cell death
 Conversion of procaspases
• Chymotrypsinogen Activation
○ Synthesised by pancreas
 Acinar cells
 Membrane bound granules
○ Single polypeptide chain
 No enzymatic activity
○ Converted to pi-chymotrypsin
 Arg15-Ile16 cleaved (by trypsin)
□ N-terminal Ile16 turns inwards, forms ionic bond with Asp194
□ Electrostatic changes causes met192 to move to the surface
□ S1 pocket formed (specificity)
□ Shifts form oxyanion hole
○ Pi-chymotrypsin acts on itself
 Dipeptides (146 and 149, 13 and 16)
○ Results
 Fully activated alpha-chymotrypsin
 Three polypeptide chains linked by disulphide bridges

○

• Common activators
○ Uidenum - concurrent activation
○ Digestion of proteins and blood clotting
 Switching numerous enzymes/proteins simultaneously
○ Common activator
 Coordinate control
○ Food enters duodenum
 Enteropeptidase
 Hydrolysis lys-Ile bond in trypsinogen
○ Pancreatic trypsin inhibitor
 Zymogen activation irreversible
 Lies in active site
□ S1 pocket
□ Maximum inhibition
 Other inhibitors
□ Inhibit common activator
□ Rapid enzyme turnover
 Tight binding of inhibitor
Proteins ^M Enzymes Page 48

 Tight binding of inhibitor
□ Haemorrhaging
□ Tissue necrosis
□ Pancreatitis

Proteins ^M Enzymes Page 49

Protein Folding
10 November 2015

10:06

Primary Structure of Bovine Insulin

•
•
•
•
•
•
•

Secreted in beta-cells in islets of Langerhans
Signal of high blood glucose
2 chains
Linked by disulphide bonds
Secretion directed by signal peptide
Extensively processed in the Golgi
Domains: Glyceralderhyde-3-phosphate dehydrogenase
○ G-3-P binding
○ NAD+ Binding
○ Large polypeptides >200 amino acids divide into domains
○ Each domain is distinct, with defined combination of secondary elements
○ Domains linked by loops

Christian B
...
, Christian Anfinsen
○ 1957 Science 125 691-692
○ Reductive cleavage of disulphide bridges in ribonuclease
 Ribonuclease A
 Hydrolyses RNA to ribonucleotides
 Tight core of beta-sheets
 4 disulphide bridges
○ Result
 8M urea disrupts H bonds and hydrophobic interaction
 Beta-mercaptoethanol (reducing agent) disrupts disulphide bonds



□ If urea is and BEM are removed at the same time the native state is achieved
□ If BEM is removed first disulphide bridges form incorrectly
 Can be renatured by addition of trace amounts of BEM
 Anfinsen's Thermodynamic Hypothesis
Proteins ^M Enzymes Page 50

 Anfinsen's Thermodynamic Hypothesis
□ Each unique 1o structure is key for a defined final conformation
□ 1o structure determines higher order protein assembly
○ Anfinsen's work showed that proteins can adopt their native conformation spontaneously
 Sequence determine structure
Forces driving protein folding
1
...
1 kJ/mole), but proteins have many atoms so cumulative effect is important
2
...

3
...
Hydrophobic interactions – crucial in protein folding, hydrophobic core, “oily centre”
...
Disulphide bonds (some proteins only, dependent on intra/extracellular environment
...
Small chaperones <300kDa
○ Grabs exposed hydrophobic regions
○ Bind and release
○ Prevents aggregation
2
...
coli, highly conserved
 DnaJ binds to unfolded protein then to DnaK
 DnaJ stimulates ATP hydrolysis by DnaK
 GrpE stimulates ADP release (Bacteria)
• Chaperonins
○ GroEL
 Hsp60 - 14 protein complex
 Binds ATP/Adp
 2 halves - 2 cycles at the same time
 Anfinsen Cage
○ GroES cochaperone - 7 subunits in ring matching GroEL
 Forms cap and enlarges cavity through conformational changes to GroEL

○

• Hsp90 - steroid hormone receptors
Proteins ^M Enzymes Page 54

• Hsp90 - steroid hormone receptors
○ Regulatory functions
○ Increased during stress
○ Cytosolic and ER forms 82-94kDa
○ Reversible binding with target acts as on/off switch
○ Regulates wide range of functions

Proteins ^M Enzymes Page 55

Protein Targeting
17 November 2015

10:28

• Problem of protein localisation
○ Eukaryotic cell contains 5x109 proteins
○ 105 different proteins
○ Different proteins in different organelles
○ Different 1/2 lives
○ Must be targeted throughout cell
Pulse-chase labelling
• Palade 1975
• Irradiate proteins and trace through secretory pathway
• Proteins for export always synthesised on ER polysomes
○ Never completely form in cytosol
• Palade Pathway through ER, Golgi, Vesicles, Release

•

•

• Endoplasmic reticulum stacks of flattened cisternae
○ RER abundant in secretory cells
○ Ribosomes must bind to ER if secretory protein, remain in cytosol if not
Signal Sequence
• Tell ribosome whether to associate with RER or remain in cytosol
• 1st sequence = signal sequence
○ Hydrophobic
• Cleavage site - signal sequence removed
Microsomes
Proteins ^M Enzymes Page 56

Microsomes
• Pulp up ER - breaks into smaller vesicles
• Proteins within microsomes won't be digested by protease addition
• Add detergent - permeable membranes - proteases can digest proteins
• Translocation coupled to translation
○ Cell-free protein synthesis
 Contains ribosomes
 RNAse to remove RNA
○ Can add RNA and produce protein of choice
○ Give RNA for secretory protein
 Complete protein will not move to microsomes added afterwards
 Signal sequence not enough to determine
○ If microsomes present during translation, ribosome moves and protein ends up in
microsome (with signal sequence removed)
Signal Recognition Particle
• SRP
• Lots of proteins
• RNA backbone
Translocation into ER
• SRP binds signal sequence
• SRP receptor in membrane
• Translocon through membrane
• Signal peptidase inside membrane cleaves signal sequence
• GTP driven
• Chaperones inside ER
○ BiP binding protein - bind and release mechanism

Topology of Membrane Proteins
• Type I
○ Glycoprotein, insulin receptor, growth hormone receptor
○ Same mechanism
 Transmembrane helix stop transfer anchor
 Hydrophobic
 Shuts down translocon
 Holds protein within membrane
○ NH3+ terminal inside, COO- cytosolic

Proteins ^M Enzymes Page 57

○

• Type II
○ Asialoglycoprotein receptor, transferin receptor
○ COO- inside, NH3+ outside
○ Enter through translocon
○ Signal peptide within sequence - signal anchor
○ Anchored within membrane and extruded sideways

○

• Type IV
○ Multiple loops/helices through membrane
○ COO- or NH4+ on either side

○

• Determined by location and order of stop transfer, signal, anchor segments
• Hydrophobic (Leucine L, Isoleucine, Valine V)
○ Hydrophobicity plot - +ve = more hydrophobic
Mitochondrial Uptake
• Proteins synthesised in cytosol
○ Uptaken if mitochondria then added (unlike ER)
• Translocons at each membrane
○ Translocons conjugate to 'skip' intermembrane space
○ Chaperone proteins within membrane

Proteins ^M Enzymes Page 58

•

Chloroplast Targeting
• 3 membranes
• Stromal import signal cleaves
• Thylakoid targeting sequence
○ Chloroplast SRP
 Similar to ER insertion
○ Or twin arginine transporter
 Different to all other translocons
 More like sphincter - monomer recruited or shed from membrane to change size
 Allow proteins of various sizes through - already folded
□ Protein must be folded around metal ions to enter thylakoid
□ Twin arganine (R) recognised - sphincter dilates as much as needed
□ Signal removed inside thylakoid

○

Proteins ^M Enzymes Page 59

Bacterial Translocation
• Sec dependent
• Translocon pumps through
• Sequence cleaved
• Lipoproteins
○ Type II signal sequence
○ Lipid groups added
○ Sequence cleaved
• Type III Sequences
○ Pushes through plasma membranes
○ Similar to flagellum
○ Push proteins down core

○

Prediction of Localisation/Targeting
• Known sequences can be used to predict
Protein Modification
• Post translational
• Normal coincident with targeting
• Only proteins with disulphide bonds are secretory (ER, Golgi)
• Ubiquitination
• Pyrrolysine and selenocysteine
• Phosphate groups
• Glycose
• Acyl groups
• Lots of important functions
○ Carboxylated glutamate important in clotting
○ Failure to g-carboxylate due to vitamin K deficiency
• Hydroxylation stabilises collagen fibres
• Sugar coating
○ Recognition sites
○ Shield protein surface - protection from protease and non-specific interaction
○ Increased solubility - less aggregation of new glycoproteins
• N-linked glycoprotein sugars
○ Core created in cytosol
○ Flipase flips through membrane
 Hydrophilic sugars, mechanism not understood
○ Further sugars added in ER
○ Some sugars act as timers
 Fall off, cell recognises degraded cells
• Spontaneous glycosylation
Proteins ^M Enzymes Page 60

• Spontaneous glycosylation
○ Accumulate sugar
○ More likely with age
GPI Anchor
• Amphipathic molecule
○ Hydrophobic and polar regions
• Made independently of protein in membrane
• Protein enters into lumen
○ Signal sequence cleaves and protein bind to anchor

Proteins ^M Enzymes Page 61

Experimenting with Proteins
24 November 2015

10:06

Proteins ^M Enzymes Page 62

Solving Structures
24 November 2015

10:06

Protein Structure Determination
• 2 main methods
○ NMR spectroscopy
○ X-ray crystallography
• x-ray crystallography requires protein to be crystalline
○ Very difficult
○ Most proteins don't crystallise
○ 39,000 structures mapped
• NMR can be performed on proteins in solution
○ Data analysis very hard
○ 6,000 structures mapped
How to determine structure
• Primary methods
○ x-ray crystallography
○ NMR spectroscopy
○ Cryo-electron microscopy
• Secondary methods
○ Circular dichroism
○ Fluorescence
○ Neutron scattering
Cryo-Electron Microscopy
• Sample flash frozen - liquid ethane
• Solvent not given time to form ice crystals
○ Obtain snapshot of sample in solution
• 2 approaches
○ 2D crystals
 2D layer forms
 Tilted and snapshots taken
 3D image built up from different angles
○ Image reconstruction
 Proteins don't always form flat layer - random aggregations
 Many photos taken at different angles
 Common features used to identify the same regions
 Can be wrong, giving incorrect proteins
How Accurate are the structures?
• Resolution of structure
○ Measure of how many data collected
• More data - greater ratio of observations to number of atomic coordinates to be determined in principle the greater accuracy
• Resolution in Angstroms
○ Lower number greater accuracy and level of atomic detail
• Hydrogen atoms visible at 1
...
5 Å

2Å

1
...


confirmations

Proteins ^M Enzymes Page 63

confirmations

Orientation of peptide planes

-

-

Fair

Good

Very good

Protein hydrogen atoms visible?

-

-

-

-

Very good

• NMR and x-ray crystallography <1Å-5Å
• Cryo-EM >5Å, usually ~10Å
X-Ray

NMR

Cryo-EM

Sample Size

Almost all

< 40kDa

Sample
Composition

Non-aggregated, crystalline,
not good with floppy or
disordered structures

Pure but in solution specialised Pure, frozen in solution
...


Liganded

Sometimes

Yes, arguably a better way to
study interactions

Yes, although small molecule
ligands are unlikely to be seen
...


> 5Å, usually higher (10Å - 20Å
...


<1 Å - 5Å

Circular Dichroism
• UV radiation
○ Chiral molecules/structures
• UV polarised
○ 2 circular waves
○ Rapid alternation
• Difference in absorption measurable
○ 200nm
○ Structures
• Deconvolute spectra
○ Fractions of helix, sheet, and 'coil'

Proteins ^M Enzymes Page 64

Handling Proteins
24 November 2015

10:06

Extraction/Purification
• Source material/tissue
○ Often limited in amount
○ Yield important
• Extraction of crude protein
○ Physical release of protein from cells
○ Time consuming and harsh
• Protect from denaturation
○ Extremes of heat and pH
○ Keep on ice (4oC)
○ Extraction buffer
• Protect from proteases
○ Protease inhibitors
• Physical separation
○ Relies on properties
○ Solubility, size, activity, stability (pH/oC), isoelectric point
Ammonium sulphate precipitation, reverse phase HPLC Solubility
Isoelectric focussing, ion exchange chromatography

○ Gel filtration, size exclusion chromatography, PAGE

pI/polarity

Size

Heat denaturation

Stability

Affinity chromatography, zymography

Activity

• Precipitation
○ Alter nature of a solution
 Salt
 Organic solvent
 Changing pH
○ Low Salt
 Protein solubility increases
 Salting-in
○ High salt
 Protein solubility decreases
 Salting-out
○ Addition of an organic solvent
 e
...
acetone
 Decrease of dielectric constant
 Decrease in protein solubility
 Denaturation in some case
○ Changing pH
 Related to functional groups
 Isoelectric point crucial
 Trichloroacetic acid (TCA)
 Will probably denature

Normal Phase/Reverse Phase Chromatography
• RP
○ Resin in column
 Stationary phase
 Non-polar
○ Solvent
 Mobile phase
Proteins ^M Enzymes Page 65

 Mobile phase
 Polar
 Contains proteins
○ Non-polar protein favours stationary phase
○ Polar washes out in solvent faster
○ Less polar solvent stepping - different proteins washed out

○

• NP
○ Non-polar mobile phase
○ Polar stationary phase
Isoelectric Point
• pH gradient
• Current run through
• Proteins move according to charge
○ Positively charged move to negative end
• pH alters charge of proteins
• At isoelectric point no net charge, no movement through gel

•

Ion Exchange Chromatography
• Anion exchange
• Immobilised cation surface
• Mix of amino acids carried through in solution
• Negative residues immobilised on surface
• Positive residues carried through with solution
• Can change pH to alter charge and release
• Can use positive resin

Proteins ^M Enzymes Page 66

•

Size-Exclusion Chromatography
• Small proteins interacts with beads
• Large proteins don't, and pass through faster
• In complex mixtures of sizes
○ Size exclusion makes mixtures less complex

Differential Centrifugation

Affinity Chromatography
• Antibodies on column
• Appropriate proteins bind
• Elute unbound
• Only protein of interest left

Proteins ^M Enzymes Page 67

• Can have enzyme bound to column
○ Glutathione (GSH)
○ Enzyme has affinity for ligand
○ Can elute with the same enzyme
Confirming it's Worked
• Quantify the purification
○ Total amount of protein (start)
○ Total amount of target protein (end)
• Total protein
○ Assays
○ Lowry's, bradford, Absorbance280
• Target protein
○ Specific assay
○ Antibodies
• Yield = % target protein remaining
• Enrichment = increase in proportion of target within membrane
○ 1/20h --> 1/5, enrichment of 4
Combined With Genetics
• 6His binds to nickel
○ Engineer a gene to make a 6His-tagged protein
• Allows affinity purification
○ Immobilised metal affinity chromatography IMAC
Purity
• Proportion of mixture
• Contaminants
• Polyenylamide gel electrophoresis
○ PAGE
○ Cheap and easy
○ SDS gives -ve charge
○ Proteins migrate
○ Based on size
• Can be 2D process
○ Separate by pI
○ Run SDS PAGE
○ If proteins same size PAGE shows 1 band
 2D shows them as separate

Proteins ^M Enzymes Page 68

○

2D Staining and Analysis
• Coomassie >75ng
• Silver staining >5ng
• Fluorescent staining >5ng
Zymography
• Impregnate gel with substrate
• Run gel
• Incubate at appropriate temp (e
...
37oC)
• Bands of clearing
○ Staining coomassie blue
○ Active proteins digest

•

Quantifying
• Software identifies and compares spots
Western Blotting
• Run gel
• Transfer to membrane
• Attach antibodies
○ Target protein
• 2o antibdy with enzyme
○ Colour change

Proteins ^M Enzymes Page 69

•

Proteins ^M Enzymes Page 70

Identifying Proteins
25 November 2015

12:10

Mass Spectrometry
• 2 common approaches
○ Peptide mass fingerprinting
○ Tandem sequencing
• Proteomics
○ 2D gel
○ Protein of interest identified
○ Chop up with trypsin
○ Put peptides into MS
○ Database finds protein from m/z
Peptide Mass Fingerprinting
• Peptide fragments
• Separated by mass

•

Tandem Sequencing
• Individual peptide fragments
○ 1 through
• Interact with inert gas - fragments
○ Separate and detect
• Compare with database
• Fragment at different points along peptide
• B or Y
• Gives sequence information and masses

Proteins ^M Enzymes Page 71

•

Proteins ^M Enzymes Page 72

Researching Proteins
01 December 2015

10:03

Proteins ^M Enzymes Page 73

Russ Morphew - FhGST-S1
01 December 2015

10:03

Fasciola hepatica Glutathione Transferase - Sigma class protein 1
Liver Fluke
• F
...
1-50
...
waste
○ Source of damage?
• Eggs
• Surface
○ Outer coat
○ Protection
○ Antibodies
• Drugs
○ How they work
○ How new drugs work
○ New vaccines focussed on targets
GSTs
• Glutathione transferase
• Phase II detoxification
I
...
Conjugation - adhere molecules covalently onto xenomolecules, more water soluble
III
...
a
...
Sigma class not identified before
...
coli - expressed sigma GST
○ Purified
• In situ expression
○ Got antibodies by infecting rabbits
○ 1D gel, probed with anti-GST antibodies
 Heavily expressed in eggs and juvenile, present in adult and adult excretion
Immuno-localisation
• Liver and bile ducts
• Testes free of protein
• Surface free (but near)
• Heavily in ovaries/eggs
Function?
• Activity rFhGST-S1
○ Model system substrates

○

○ Lipid peroxide metabolism
 Detoxification of worm
○ Inhibition (conc

---

Title: Proteins and Enzymes
Description: Second year Proteins and Enzymes module, taught by Russ Morphew. Covered protein structures, thermodynamics, catalysts, folding, targeting, and experimenting.

Buy These NotesPreview