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Proteins S-notes
Amino Acids
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Life fights entropy – converts energy into information
o Builds does  metabolism
Catabolism: breaking down stuff
o Conversion of large complex molecules to smaller molecules
o Produces ATP
Anabolism: synthesis of large molecules from smaller molecules
o Requires input of energy (ATP)
Amino acids – building blocks of proteins
o Proteins are polypeptides
o Amino acids are monomers
 Protein’s form and function – determined by its amino acids
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Anatomy of amino acid
o Structure of common amino acids
o Small organic molecules containing amino group (-NH3+), carboxyl group (-COO-), and
side chain of variable structure (R group)
 R determines amino acid type
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o Optical isomers (L and D forms)
 C – chiral
 L & D isomers are mirror images
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Only L isomers are used in proteins (L IS LIFE)
20 standard amino acids
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Classify amino acids into non-polar, polar and charged
Discuss how side chain properties affect interactions
o Non Polar Amino Acids
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 Hydrophobic side chains: van der Waals interactions
 Mainly hydrocarbon side chains
 Don’t react well with water; Like Lipids <3
o Polar Amino Acids
 Side chains with polar bonds: H-bonds
 R group contains electronegative atom – hoards electrons
 Interacts with water
 Stabilises structure
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o Charged Amino Acids
 Charged side chains: H-bonds & electrostatic interactions
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o Hydrophilic
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o AA – negatively charged; acidic side chains
Discuss how pH affects side chain properties
o Ionisation states depend on pH
o Microenvironment determines reactivity
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o Aa  dipolar form in neutral solution; amino group – protonated and carboxyl group –
deprotonated
o Acid solution – amino group is protonated and carboxyl isn’t dissociated
o As pH is raised, carboxylic acid is first group to give up proton
o Dipolar form persists until pH approaches 9, protonated amino group loses proton
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Essential and non-essential
o 9 amino acids cannot be made by humans
 Must get from diet  Essential
o Humans cannot synthesis ‘essential’ amino acids
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Peptide bonds and polypeptide formation
o Amino acids – joined by peptide bonds to form proteins  polypeptides
o Explain process of peptide bond and formation and ‘read’ a polypeptide
o Peptide bond – formed by dehydration reaction between carboxyl group of one amino
acid and amino group of the next
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o Contains Amino (N) terminus and carboxyl (C) terminus
o Amino acids added in turn to carboxyl terminus
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o Polypeptides have polarity and can be read in sequence.
Protein Structure
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Diverse functions and key properties
o Proteins – most versatile macromolecules in living systems
o Have diverse biological functions
 Structural (scaffolding of cell)
 Catalytic (enzymes)
 Signal transduction (hormones)
 Transport
 Control of growth and differentiation (growth factors)
 Movement
 Immune protection
o Proteins are polypeptides
Key Properties
o Proteins are linear polymers build of monomer units  amino acids
o R group  determines property
o Contain wide range of functional groups
o Can interact with (lots of things) low molecular weight compounds, with one another and
with other biological macromolecules
o Each type has unique 3D shape/ conformation
o Quite rigid (structural elements in cells), flexible (hinges, springs & levers) or are even
unstructured
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Describe the 4 levels of protein structure
Primary structure (sequence)
o Amino acids linked by peptide bonds to form polypeptide chains
o Unique sequence of amino acids
o Like order of letters in long word
o Sequences determined by inherited genetic information
o Peptide bonds are planar
 Tendency to drag e- away from double bonds
 Fluctuating double bond  stops rotation  fixed part
 Pair of linked acids 6 atoms (Ca, C, O, N, H and Ca) lie in a plane
 Mostly trans not cis  steric clashes
 Two side chains close  not enough space  steric clashes
 Other bonds – free to rotate
 Flexible peptide back bone (but there are constraints)
 Polypeptide chain has directionality – because ends are different
 A-amino group at one end, and α- carboxyl at other
 Sequence written starting with amino-terminal residue
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Secondary Structure
o Parts of polypeptide chains fold into regular structures such as Alpha Helix, Beta Sheet
and Turns and Loops
o Conformation adopted by local regions of polypeptide
o 2 elements: α-helix and β-strand
 Depend on hydrogen bonding between C=O and N-H groups along polypeptide
backbone (no side chain interactions)
 H bonds stabilises it
o Turns and Loops
 Regions that allow changes in direction (reversals) of polypeptide chain
o α-helix
 Right handed (clockwise)
 Like DNA but only single helix
 Intrachain hydrogen-bonds  H bonds in same polypeptide chain
 Backbone – H bond  inside and stabilises
 R side chains on outside
 CO group of residue i forms H bond with NH group of reside i + 4
o β-sheets
o Antiparallel β-strand
 Adjacent β strands run in opposite directions
 H-bonds between N-H and C=O groups between single pairs of amino acids
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o Parallel β-sheets
 Adjacent β strands run in same direction
 From N  C
 H-bonds between N-H and C=O groups connect each amino acid to two others
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o Mixed β sheets
 Parallel and antiparallel β strands
 Backbone interaction; not R groups
 Side chains alternate above and below plane of strand
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Tertiary structure (folding)
o Overall conformation of fully folded protein
o Involves interactions between side chains of amino acids
o Final fully folded form
 Irregular contortions created by interactions between side chains of various amino
acids
o Driven by side chain interactions, (not back bone)
o Hydrophobic Effect
 Polypeptide chain folds so its hydrophobic side chains are buried and its
polar/charged side chains are on/face towards surface
o Turns and Loops
 Keep globular state
 Usually on surface of proteins: interactions determined by R groups
 Hydrophilic
 Flexible
 More free to evolve and different substitutions
o Transition from disordered mixture of unfolded molecules to relatively uniform solution
of folded protein
 Overall reduction in free energy  more stable
o Stabilisation of Tertiary Structure
o Weak interactions contributing to tertiary structure of protein:
 Hydrophobic interactions
 Hydrogen Bonds
 Ionic/electrostatic bonds
 Although weak bonds, their cumulative effect helps give protein specific shape
o Disulfide bridges
 Covalent bonds between side chains of 2 cysteines
 Depends on redox state – determines propensity to form
 Disulfide bonds are reversible
 Reducing (Intracellular) - no tendency to form S-H bonds
 Oxidising (Extracellular) – form S-H bonds
o Sometimes protein folding needs help
 Chaperones assist proteins to (re)fold
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Quaternary structure
o Polypeptide chains can assemble into multi-subunit structures
o Proteins build of multiple domains
 Domains reused in different contexts
 Fold independently
 Separate bits maintain structure
o Proteins consisting of more than one polypeptide chain
 Each individual polypeptide  subunit
 Subunits usu held together by non-covalent bonds
 Can have disulphide bridges between subunits (stabilises structure)
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Discuss how protein function derives from subunit and domain composition
o Sequence determines structure
o Different amino acid sequence will produce different conformation that will have
different biological function
o Structural similarity =/= sequence similarity
o Domains  have clearly identifiable functions
o Functions are often ligand-binding
o Functional classification of proteins
 Structural proteins e.g. collagen
 Contractile proteins e.g. muscle proteins
 Blood proteins e.g. haemoglobin
 Hormones e.g. insulin
 Enzymes e.g. biological catalysts
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Denaturation of Protein
o High T, mechanical forces and chemical treatments
 Can denature protein, causing it to lose its conformation and ability to function
 Disrupts weak interactions
o Denaturation – loss of biological activity
o Protein can be re-natured when chemical and physical aspects of environment are restored
to normal  favourable conditions
o Can also be irreversible
Enzymes and Catalysis
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What is an enzyme?
o Catalysts of biological systems
o Typically proteins
o Bind variety of molecules but each enzymes binds only to a couple of molecules, with
high specificity
o E.g. Lysozyme protein – enzyme that breaks down bacterial cell walls – in tears
Order Nomenclature
o Common names e.g. trypsin breaks large protein into smaller peptides
 Pepain  tenderise meat, breaks down collagen, found in papaya
o Conventionally, enzymes named for substrates and reactions they catalyse
 Suffix ‘-ase’ is added
 ATPase – breaks down ATP
 ATP synthase – synthesises ATP
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What is a substrate?
o Reactants in enzyme mediated reactions
o Converted into new products
o Products can themselves be substrates for new reactions
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Substrate specificity
o Determined by protein 3D structure and structure of active site
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o Structure of active sites – complicated
 Polypeptide chain folded into 3D enzyme molecule
 Substrate molecule bound to active site of enzyme molecule
 Active site – small part of protein
 Are clefts or crevices
 Binding between enzyme and substrate is from weak forces
o Van der Waals forces, H bonds, ionic forces
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What is catalysis?
o Acceleration of reaction rate of chemical reaction by means of catalyst
o Does not make new chemical reactions possible
o Only speeds up existing reactions
 Catalyst not consumed by reaction and can catalyse same reaction many times
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Free energy and activation energy
o Free energy: Amount of energy in system which can be converted into work
 Enzyme substrates and products have free energy
o Differences in free energy between substrates and products (∆G) account for reaction
equilibrium
o If Product lower free energy than substrate, reaction can occur spontaneously (but
reaction rate may be very slow)
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o Activation energy – energy hurdle that must be overcome for reaction to take place
 Independent of reaction’s change in free energy
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o Enzymes lower reaction activation energy
 Accelerate chemical reaction by providing lower energy pathway between
substrates and reaction products
 Lower activation energy of reaction – increases likelihood that reaction will
happen
o Enzymes don’t alter reaction equilibrium
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o They lower activation energy by assisting formation of one or more reaction
intermediates/ transition states which cannot be formed without catalyst
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Transition states in catalysis
o Transition states – seldom seen in nature
 They occur too rapidly
o We know they exist as
 Enzymes can be inhibited by transition-state analogs
Induced fit model of catalysis
o Lock and key’ models – oversimplification
o Induced fit model suggests
 Binding of substrate changes shape of active site
 Change in active site favours transition state
 Products are produced and released
 Enzyme resumes normal shape after catalytic event
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Optimal conditions for catalysis
o pH must be correct
 most enzymes have optimal activity at physiological pH (7.2-7.3)
o Temperature must be correct
 37oC for mammals
 41oC for birds
 Extreme temps for extremophiles
o Co-factors must be present
 Vitamin associated cofactors
 Metals (e.g. zinc, potassium)
Enzyme Kinetics and Regulation
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What is kinetics?
o Rate at which an enzyme works
Why study enzyme kinetics?
o To understand enzyme catalytic power
 When and why it works best
 Factors that affect activity (inhibitors)
o Important for biology
o Of economic importance for industry
o Central to pharmaceutical design
Enzyme reaction velocity
o Affected by Substrate concentration
o Measurements made for fixed amount of enzyme
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Michaelis-Menten value. KM
o KM – substrate concentration at which enzyme reaction velocity is half its maximal rate
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o Enzymes have different turnover values  number of molecules of substrate converted to
produce per second
o Turnover ∝ Vmax
o KM – substrate concentration required for significant catalysis to occur
 Value related to rate constants of steps of catalysis
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Michaelis-Menten equation
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= reaction rate
= maximum rate
= substrate concentration
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o Case 1: when [S] is very low and smaller than
, V0 ∝ [S]
o Case 2: when [S] is very large and greater than KM, V0 = VMax
o PROBLEMS
 VMax is approached asymptotically, which is difficult to measure
 So KM diff to measure:
Lineweaver-Burk plots
o DOUBLE RECIPROCAL OF MICHAELIS-MENTEN PLOTS
o Plot of 1/V0 versus 1/[S]
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Enzyme inhibition
o No enzyme inhibition
 When no inhibitors are present, enzymes function as:
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o Competitive Inhibition
 Inhibitor molecules bind active site of enzyme
 Inhibitor competes with binding of substrate, thus reducing rate of catalysis
 Degree of inhibition depends on concentration of substrate and inhibitor
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Affects enzyme kinetics
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Same VMax can be reached with higher [S]
Inhibits reaction
Apparent KM is higher
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 Inhibitor (I) can =bind to enzyme (E) to form EI complex
o Non-Competitive Inhibition
 Inhibitor molecules bind to another site of enzyme
 Alters structure of active site, affecting capacity of enzyme to convert substrate to
product
 Does NOT compete with binding of substrate
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Lower VMax seen
KM remains unchanged
Inhibitor stops proportion of all enzymes from converting substrate to product
Concentration of functional enzyme has been effectively lower but can work at
same velocity
Increases in substrate concentration has no effect
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Allosteric inhibition
o Allosteric enzymes contain distinct regulatory and active sites
o Small molecules can bind to regulatory sites
o Cause conformational changes that reduce catalysis at active site
o SOME enzymes show feedback inhibition where products formed are their own inhibitors
o Where feedback inhibition is found, inhibition is often to first enzyme in pathway
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o Allosteric Enzymes do NOT show Michaelis-Menten kinetics
o Allosteric enzymes show sigmoidal kinetics (resembles an S)
o Represent superimposition of 2 curves (one for enzyme with low KM and other with high
KM
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