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Enzyme Kinetics

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Title: Enzyme Kinetics


1
Enzyme Kinetics Protein Folding9/7/2004
2
Protein folding is one of the great unsolved
problems of science Alan Fersht
3
protein folding can be seen as a connection
between the genome (sequence) and what the
proteins actually do (their function).
4
Protein folding problem
  • Prediction of three dimensional structure from
    its amino acid sequence
  • Translate Linear DNA Sequence data to spatial
    information

5
Why solve the folding problem?
  • Acquisition of sequence data relatively quick
  • Acquisition of experimental structural
    information slow
  • Limited to proteins that crystallize or stable in
    solution for NMR

6
Protein folding dynamics
Electrostatics, hydrogen bonds and van der Waals
forces hold a protein together. Hydrophobic
effects force global protein conformation. Peptide
chains can be cross-linked by disulfides, Zinc,
heme or other liganding compounds. Zinc has a
complete d orbital , one stable oxidation state
and forms ligands with sulfur, nitrogen and
oxygen. Proteins refold very rapidly and
generally in only one stable conformation.
7
The sequence contains all the information to
specify 3-D structure
8
Random search and the Levinthal paradox
  • The initial stages of folding must be nearly
    random, but if the entire process was a random
    search it would require too much time. Consider a
    100 residue protein. If each residue is
    considered to have just 3 possible conformations
    the total number of conformations of the protein
    is 3100. Conformational changes occur on a time
    scale of 10-13 seconds i.e. the time required to
    sample all possible conformations would be 3100 x
    10-13 seconds which is about 1027 years. Even if
    a significant proportion of these conformations
    are sterically disallowed the folding time would
    still be astronomical. Proteins are known to fold
    on a time scale of seconds to minutes and hence
    energy barriers probably cause the protein to
    fold along a definite pathway.

9
Energy profiles during Protein Folding
10
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11
Physical nature of protein folding
  • Denatured protein makes many interactions with
    the solvent water
  • During folding transition exchanges these
    non-covalent interactions with others it makes
    with itself

12
What happens if proteins don't fold correctly?
  • Diseases such as Alzheimer's disease, cystic
    fibrosis, Mad Cow disease, an inherited form of
    emphysema, and even many cancers are believed to
    result from protein misfolding

13
Protein folding is a balance of forces
  • Proteins are only marginally stable
  • Free energies of unfolding 5-15 kcal/mol
  • The protein fold depends on the summation of all
    interaction energies between any two individual
    atoms in the native state
  • Also depends on interactions that individual
    atoms make with water in the denatured state

14
Protein denaturation
  • Can be denatured depending on chemical
    environment
  • Heat
  • Chemical denaturant
  • pH
  • High pressure

15
Thermodynamics of unfolding
  • Denatured state has a high configurational
    entropy
  • S k ln W
  • Where W is the number of accessible states
  • K is the Boltzmann constant
  • Native state confirmationally restricted
  • Loss of entropy balanced by a gain in enthalpy

16
Entropy and enthaply of water must be added
  • The contribution of water has two important
    consequences
  • Entropy of release of water upon folding
  • The specific heat of unfolding (?Cp)
  • icebergs of solvent around exposed hydrophobics
  • Weakly structured regions in the denatured state

17
The hydrophobic effect
18
High ?Cp changes enthalpy significantly with
temperature
  • For a two state reversible transition
  • ?HD-N(T2) ?HD-N(T1) ?Cp(T2 T1)
  • As ?Cp is positive the enthalpy becomes more
    positive
  • i.e. favors the native state

19
High ?Cp changes entropy with temperature
  • For a two state reversible transition
  • ?SD-N(T2) ?SD-N(T1) ?CpT2 / T1
  • As ?Cp is positive the entropy becomes more
    positive
  • i.e. favors the denatured state

20
Free energy of unfolding
  • For
  • ?GD-N ?HD-N - T?SD-N
  • Gives
  • ?GD-N(T2) ?HD-N(T1) ?Cp(T2 T1)-
    T2(?SD-N(T1) ?CpT2 / T1)
  • As temperature increases T?SD-N increases and
    causes the protein to unfold

21
Cold unfolding
  • Due to the high value of ?Cp
  • Lowering the temperature lowers the enthalpy
    decreases
  • Tc T2m / (Tm 2(?HD-N / ?Cp)
  • i.e. Tm 2 (?HD-N ) / ?Cp

22
Measuring thermal denaturation
23
Solvent denaturation
  • Guanidinium chloride (GdmCl) H2NC(NH2)2.Cl-
  • Urea H2NCONH2
  • Solublize all constitutive parts of a protein
  • Free energy transfer from water to denaturant
    solutions is linearly dependent on the
    concentration of the denaturant
  • Thus free energy is given by
  • ?GD-N ?HD-N - T?SD-N

24
Solvent denaturation continued
  • Thus free energy is given by
  • ?GD-N ?GH2OD-N - mD-N denaturant

25
Acid - Base denaturation
  • Most proteins denature at extremes of pH
  • Primarily due to perturbed pKas of buried
    groups
  • e.g. buried salt bridges

26
Two state transitions
  • Proteins have a folded (N) and unfolded (D) state
  • May have an intermediate state (I)
  • Many proteins undergo a simple two state
    transition
  • D ltgt N

27
Folding of a 20-mer poly Ala
28
Unfolding of the DNA Binding Domain of HIV
Integrase
29
Two state transitions in multi-state reactions
30
Rate determining steps
31
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32
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33
Theories of protein folding
  • N-terminal folding
  • Hydrophobic collapse
  • The framework model
  • Directed folding
  • Proline cis-trans isomerisation
  • Nucleation condensation

34
Molecular Chaperones
  • Three dimensional structure encoded in sequence
  • in vivo versus in vitro folding
  • Many obstacles to folding
  • Dlt----gtN
  • ?
  • Ag

35
Molecular Chaperone Function
  • Disulfide isomerases
  • Peptidyl-prolyl isomerases (cyclophilin, FK506)
  • Bind the denatured state formed on ribozome
  • Heat shock proteins Hsp (DnaK)
  • Protein export delivery SecB

36
What happens if proteins don't fold correctly?
  • Diseases such as Alzheimer's disease, cystic
    fibrosis, Mad Cow disease, an inherited form of
    emphysema, and even many cancers are believed to
    result from protein misfolding

37
GroEL
38
GroEL (HSP60 Cpn60)
  • Member of the Hsp60 class of chaperones
  • Essential for growth of E. Coli cells
  • Successful folding coupled in vivo to ATP
    hydrolysis
  • Some substrates work without ATP in vitro
  • 14 identical subunits each 57 kDa
  • Forms a cylinder
  • Binds GroES

39
GroEL is allosteric
  • Weak and tight binding states
  • Undergoes a series of conformation changes upon
    binding ligands
  • Hydrolysis of ATP follows classic sigmoidal
    kinetics

40
Sigmoidal Kinetics
  • Positive cooperativity
  • Multiple binding sites

41
Allosteric nature of GroEL
42
GroEL changes affinity for denatured proteins
  • GroEL binds tightly
  • GroEL/GroES complex much more weakly

43
GroEL has unfolding activity
  • Annealing mechanism
  • Every time the unfolded state reacts it
    partitions to give a proportion kfold/(kmisfold
    Kfold) of correctly folded state
  • Successive rounds of annealing and refolding
    decrease the amount of misfolded product

44
GroEL slows down individual steps in folding
  • GroEL14 slows barnase refolding 400 X slower
  • GroEL14/GroES7 complex slows barnase refolding 4
    fold
  • Truncation of hydrophobic sidechains leads to
    weaker binding and less retardation of folding

45
Active site of GroEL
  • Residues 191-345 form a mini chaperone
  • Flexible hydrophobic patch

46
Role of ATP hydrolysis
47
The GroEL Cycle
48
A real folding funnel
49
Amyloids
  • A last type of effect of misfolded protein
  • protein deposits in the cells as fibrils
  • A number of common diseases of old age, such as
    Alzheimer's disease fit into this category, and
    in some cases an inherited version occurs, which
    has enabled study of the defective protein

50
Known amyloidogenic peptides
  • CJD  spongiform encepalopathies  prion protein
    fragments 
  • APP  Alzheimer  beta protein fragment 1-40/43
  • HRA  hemodialysis-related amyloidosis  beta-2
    microglobin
  • PSA  primary systmatic amyloidosis 
    immunoglobulin light chain and fragments
  • SAA 1  secondary systmatic amyloidosis  serum
    amyloid A 78 residue fragment
  • FAP I  familial amyloid polyneuropathy I 
    transthyretin fragments, 50 allels
  • FAP III  familial amyloid polyneuropathy III 
    apolipoprotein A-1 fragments
  • CAA  cerebral amyloid angiopathy  cystatin C
    minus 10 residues
  • FHSA  Finnish hereditary systemic amyloidosis 
    gelsolin 71 aa fragment
  • IAPP  type II diabetes  islet amyloid
    polypeptide fragment (amylin)
  • ILA  injection-localized amyloidosis  insulin
  • CAL  medullary thyroid carcinoma  calcitonin
    fragments
  • ANF  atrial amyloidosis  atrial natriuretic
    factor
  • NNSA  non-neuropathic systemic amylodosis 
    lysozyme and fragments
  • HRA  hereditary renal amyloidosis  fibrinogen
    fragments

51
Transthyretin
  • transports thyroxin and retinol binding protein
    in the bloodstream and cerebrospinal fluid
  • senile systemic amyloidosis, which affects 
    people over 80, transtherytin forms fibrillar
    deposits in the heart. which leads to congestive
    heart failure
  • Familial amyloid polyneuropathy (FAP) affects
    much younger people causing protein deposits in
    the heart, and in many other tissues deposits
    around nerves can lead to paralysis

52
Transthyretin structure
  • tetrameric. Each monomer has two 4-stranded
    b-sheets, and a short a-helix. Anti-parallel
    beta-sheet interactions link monomers into dimers
    and a short loop from each monomer forms the main
    dimer-dimer interaction. These pairs of loops
    keep the two halves of the structure apart
    forming an internal channel.

53
Fibril structure
  • Study of the fibrils is difficult because of its
    insolubility making NMR solution studies
    impossible and they do not make good crystals
  • X-ray diffraction, indicates a pattern
    consistent with a long b-helical structure, with
    24 b-strands per turn of the b-helix.

54
Formation of proto-filaments
  • Four twisted b-helices make up a proto-filament
    (50-60A)
  • Four of these associate to form a fibril as seen
    in electron microscopy (130A)
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