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					            Enzyme Regulation:
             Globin examples


                  Andy Howard
       Introductory Biochemistry, Fall 2008
            Tuesday 4 November 2008

Biochemistry: Regulation
                              1           11/04/2008
     Hemoglobin as an
     honorary enzyme
    We’ll illustrate some of our
     understandings of regulation and
     allostery via hemoglobin and
     myoglobin. But first we need to
     finish establishing general principles
     about enzyme regulation.

11/04/2008   Biochemistry: Regulation   p. 2 of 44
    Mechanism Topics
   Regulation,                   Globins as
    concluded                      Examples
   Allostery: more                    Oxygen binding
    details                            Tertiary structure
                                       Quarternary
   Post-translational                  structure
    modification                       R and T states
   Protein-protein                    Allostery
    interactions                       Bohr effect
                                       BPG as an effector
                                       Sickle-cell anemia
11/04/2008   Biochemistry: Regulation              p. 3 of 44
     Regulation of enzymes
     (review)
   The very catalytic proficiency for which
    enzymes have evolved means that their
    activity must not be allowed to run amok
   Activity is regulated in many ways:
       Thermodynamics
       Enzyme availability
       Allostery
       Post-translational modification
       Protein-protein interactions

11/04/2008     Biochemistry: Regulation   p. 4 of 44
Kinetics of
allosteric enzymes
   Generally these don’t obey Michaelis-
    Menten kinetics
   Homotropic positive effectors produce
    sigmoidal (S-shaped) kinetics curves
    rather than hyperbolae
   This reflects the fact that the binding of
    the first substrate accelerates binding of
    second and later ones

11/04/2008   Biochemistry: Regulation    p. 5 of 44
 T  R State transitions
   Many allosteric effectors influence the
    equilibrium between two conformations
   One is typically more rigid and inactive,
    the other is more flexible and active
   The rigid one is typically called the “tight”
    or “T” state; the flexible one is called the
    “relaxed” or “R” state
   Allosteric effectors shift the equilibrium
    toward R or toward T

11/04/2008   Biochemistry: Regulation      p. 6 of 44
 MWC model
 for allostery
    Emphasizes
     symmetry and
     symmetry-breaking
     in seeing how
     subunit interactions
     give rise to
     allostery
    Can only explain
     positive
     cooperativity
11/04/2008   Biochemistry: Regulation   p. 7 of 44
 Koshland (KNF) model
    Emphasizes conformational changes from
     one state to another, induced by binding
     of effector
    Ligand binding and conformational
     transitions are distinct steps
    … so this is a sequential model for
     allosteric transitions
    Allows for negative cooperativity as well
     as positive cooperativity

11/04/2008   Biochemistry: Regulation   p. 8 of 44
      Heterotropic effectors




11/04/2008   Biochemistry: Regulation   p. 9 of 44
 Post-translational modification
   We’ve already looked at phosphorylation
   Proteolytic cleavage of the enzyme to
    activate it is another common PTM mode
   Some proteases cleave themselves
    (auto-catalysis); in other cases there’s an
    external protease involved
   Blood-clotting cascade involves a series
    of catalytic activations

11/04/2008   Biochemistry: Regulation   p. 10 of 44
 Zymogens
   As mentioned earlier, this is a term for an
    inactive form of a protein produced at the
    ribosome
   Proteolytic post-translational processing
    required for the zymogen to be converted
    to its active form
   Cleavage may happen intracellularly,
    during secretion, or extracellularly

11/04/2008   Biochemistry: Regulation   p. 11 of 44
     Blood clotting
   Seven serine proteases in cascade
   Final one (thrombin) converts fibrinogen
    to fibrin, which can aggregate to form an
    insoluble mat to prevent leakage
   Two different pathways:
        Intrinsic: blood sees injury directly
        Extrinsic: injured tissues release factors that
         stimulate process
   Come together at factor X

11/04/2008     Biochemistry: Regulation          p. 12 of 44
Cascade



 11/04/2008   Biochemistry: Regulation   p. 13 of 44
 Protein-protein interactions
   One major change in biochemistry in the last 20
    years is the increasing emphasis on protein-
    protein interactions in understanding biological
    activities
   Many proteins depend on exogenous partners
    for modulating their activity up or down
   Example: cholera toxin’s enzymatic component
    depends on interaction with human protein
    ARF6


11/04/2008   Biochemistry: Regulation       p. 14 of 44
 Globins as aids to
 understanding
    Myoglobin and hemoglobin are well-
     understood non-enzymatic proteins
     whose properties help us understand
     enzyme regulation
    Hemoglobin is described as an “honorary
     enzyme” in that it “catalyzes” the reaction
     O2(lung)  O2 (peripheral tissues)

11/04/2008   Biochemistry: Regulation   p. 15 of 44
    Setting the stage for this story
   Myoglobin is a 16kDa monomeric O2-storage
    protein found in peripheral tissues
   Has Fe-containing prosthetic group called
    heme; iron must be in Fe2+ state to bind O2
   It yields up dioxygen to various oxygen-
    requiring processes, particularly oxidative
    phosphorylation in mitochondria in rapidly
    metabolizing tissues



11/04/2008   Biochemistry: Regulation   p. 16 of 44
 Why is myoglobin needed?
    Free heme will bind O2 nicely;
     why not just rely on that?
    Protein has 3 functions:
         Immobilizes the heme group
         Discourages oxidation of Fe2+ to Fe3+
         Provides a pocket that oxygen can fit into


11/04/2008     Biochemistry: Regulation     p. 17 of 44
 Setting the stage II
    Hemoglobin (in vertebrates, at least) is a
     tetrameric, 64 kDa transport protein that
     carries oxygen from the lungs to
     peripheral tissues
    It also transports acidic CO2 the opposite
     direction
    Its allosteric properties are what we’ll
     discuss

11/04/2008   Biochemistry: Regulation   p. 18 of 44
    Structure determinations
   Myoglobin & hemoglobin were the first
    two proteins to have their 3-D structures
    determined experimentally
        Myoglobin: Kendrew, 1958
        Hemoglobin: Perutz, 1958
        Most of the experimental tools that
         crystallographers rely on were developed for
         these structure determinations
   Nobel prizes for both, 1965 (small T!)

11/04/2008     Biochemistry: Regulation       p. 19 of 44
                                                   QuickTime™ an d a

    Myoglobin structure                   TIFF (Uncompressed) decompressor
                                             are need ed to see this p icture .




   Almost entirely -helical
   8 helices, 7-26 residues each                  Sperm whale
                                               myoglobin; 1.4 Å
   Bends between helices generally short 18 kDa monomer
   Heme (ferroprotoporphyrin IX) tightly but       PDB 2JHO
    noncovalently bound in cleft between helices E&F
   Hexacoordinate iron is coordinated by 4 N atoms
    in protoporphyrin system and by a histidine side-
    chain N (his F8): fig.15.25
   Sixth coordination site is occupied by O2, H2O,
    CO, or whatever else fits into the ligand site

11/04/2008     Biochemistry: Regulation                  p. 20 of 44
    O2 binding alters myoglobin
    structure a little
   Deoxymyoglobin: Fe2+ is 0.55Å out of the
    heme plane, toward his F8, away from O2
    binding site
   Oxymyoglobin: moves toward heme
    plane—now only 0.26Å away (fig.15.26)
   This difference doesn’t matter much
    here, but it’ll matter a lot more in
    hemoglobin!

11/04/2008   Biochemistry: Regulation   p. 21 of 44
      Hemoglobin
      structure                                  QuickTime™ an d a
                                        TIFF (Uncompressed) decompressor
                                           are need ed to see this p icture .
   Four subunits, each closely
    resembling myoglobin in
    structure (less closely in
    sequence);
                                                     Human
    H helix is shorter than in Mb                    deoxyHb
   2 alpha chains,                                  PDB 2HHB
    2 beta chains                                    1.74Å
                                                     65kDa
                                                     hetero-
                                                     tetramer


11/04/2008   Biochemistry: Regulation                p. 22 of 44
    Subunit                                            QuickTime™ and a
                                              TIFF (Uncompressed) decompressor
                                                 are neede d to see this picture.




    interfaces in Hb
   Subunit interfaces are
    where many of the                     Image courtesy
    allosteric interactions               Pittsburgh
    occur                                 Supercomputing
       Strong interactions:              Center
        1 with 1 and 2,
        1 with 1 and 2
       Weaker interactions:
        1 with 2, 1 with 2

11/04/2008     Biochemistry: Regulation                      p. 23 of 44
     Subunit dynamics
   1-1 and 2-2 interfaces are solid and
    don’t change much upon O2 binding
   1-2 and 2-1 change much more:
    the subunits slide past one another by 15º
       Maximum movement of any one atom ~ 6Å
       Residues involved in sliding contacts are in
        helices C, G, H, and the G-H corner
   This can be connected to the oxygen
    binding and the movement of the iron atom
    toward the heme plane

11/04/2008     Biochemistry: Regulation        p. 24 of 44
      Conformational states
   We can describe this shift as a transition from
    one conformational state to another
   The stablest form for deoxyHb is described as a
    “tense” or T state
       Heme environment of beta chains is almost
        inaccessible because of steric hindrance
       That makes O2 binding difficult to achieve
   The stablest form for oxyHB is described as a
    “relaxed” or R state
   Accessibility of beta chains substantially
    enhanced


11/04/2008     Biochemistry: Regulation         p. 25 of 44
 Hemoglobin allostery
    Known since early 1900’s that
     hemoglobin displayed sigmoidal oxygen-
     binding kinetics
    Understood now to be a function of
     higher affinity in 2nd, 3rd, 4th chains for
     oxygen than was found in first chain
    This is classic homotropic allostery even
     though this isn’t really an enzyme


11/04/2008   Biochemistry: Regulation    p. 26 of 44
 R  T states and hemoglobin
   We visualize each Hb monomer as
    existing in either T (tight) or R (relaxed)
    states; T binds oxygen reluctantly, R
    binds it enthusiastically
   DeoxyHb is stablest in T state
   Binding of first Hb stabilizes R state in
    the other subunits, so their affinity is
    higher

11/04/2008   Biochemistry: Regulation     p. 27 of 44
  Hill coefficients
      Recognition that binding could be modeled by a
       polynomial fit to pO2
      Kinetics worked out in 1910’s: didn’t require
       protein purification, just careful in vitro
       measurements of blood extracts
      Actual equation is on next page
      Relevant parameters to determine are P50, the
       oxygen partial pressure at which half the O2-
       binding sites are filled, and n, a unitless value
       characterizing the cooperativity
      n is called the Hill coefficient.

11/04/2008    Biochemistry: Regulation         p. 28 of 44
  PO2 and fraction oxygenated
    If Y is fraction of globin that is oxygenated and
     pO2 is the partial pressure of oxygen,
     then Y/(1-Y) = (pO2 /P50)n
    P50 is a parameter corresponding to half-
     occupied hemoglobin
         work out the algebra:
         When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2.
    Note that the equation on p.496 is wrong!



11/04/2008      Biochemistry: Regulation         p. 29 of 44
 Real Hill parameters (p.496)
   Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr
        Perfect cooperativity, tetrameric protein: n =4
        No cooperativity at all would be n = 1.
   Lung pO2 ~ 100 Torr;
    peripheral tissue 10-40 Torr
   So lung has Y~0.98, periphery has Y~0.06!
   That’s a big enough difference to be functional
   If n=1, Ylung=0.79, Ytissue=0.28; not nearly as
    good a delivery vehicle!



11/04/2008      Biochemistry: Regulation           p. 30 of 44
 MWC theory
   Monod, Wyman, Changeux developed
    mathematical model describing TR
    transitions and applied it to Hb
   Accounts reasonably well for sigmoidal
    kinetics and Hill coefficient values
   Key assumption: ligand binds only to R
    state, so when it binds,
    it depletes R in the TR equilibrium,
    so that tends to make more R

11/04/2008   Biochemistry: Regulation   p. 31 of 44
 Koshland’s contribution

    Conformational changes between the two
     states are also clearly relevant to the
     discussion
    His papers from the 1970’s articulating
     the algebra of hemoglobin-binding
     kinetics are amazingly intricate


11/04/2008   Biochemistry: Regulation   p. 32 of 44
 Added complication I: pH
   Oxygen affinity is pH dependent
   That’s typical of proteins, especially those in
    which histidine is involved in the activity
    (remember it readily undergoes protonation and
    deprotonation near neutral pH)
   Bohr effect (also discovered in early 1900’s):
    lower affinity at low pH (fig. 15.33)




11/04/2008   Biochemistry: Regulation       p. 33 of 44
    How the Bohr
    effect happens
   R form has an effective pKa that
    is lower than T                          Cartoon
                                             courtesy
   One reason:                              John
       In the T state, his146 is close to   Robertus,
        asp 94. That allows the histidine    UT Austin
        pKa to be higher
       In R state, his146 is farther from
        asp 94 so its pKa is lower.


11/04/2008     Biochemistry: Regulation      p. 34 of 44
 Physiological result
 of Bohr effect
    Actively metabolizing tissues tend to
     produce lower pH
    That promotes O2 release where it’s
     needed




11/04/2008   Biochemistry: Regulation   p. 35 of 44
 CO2 also promotes
 dissociation
    High [CO2] lowers pH because it
     dissolves with the help of the enzyme
     carbonic anhydrase and dissociates:
     H2O + CO2  H2CO3  H+ + HCO3-
    Bicarbonate transported back to lungs
    When Hb gets re-oxygenated,
     bicarbonate gets converted back to
     gaseous CO2 and exhaled
11/04/2008   Biochemistry: Regulation   p. 36 of 44
 Role of carbamate

    Free amine groups in Hb react reversibly
     with CO2 to form R—NH—COO- + H+
    The negative charge on the amino
     terminus allows it to salt-bridge to Arg
     141
    This stabilizes the T (deoxy) state


11/04/2008   Biochemistry: Regulation   p. 37 of 44
                                                   Qu i ckTi me ™ an d a
                                        TIFF (Un co mp re ss e d) de co mp re ss or
                                           a re ne ed ed to se e thi s p i ctu re .



 Another allosteric
 effector                                     BPG (Wikimedia)

    2,3-bisphosphoglycerate is a heterotropic
     allosteric effector of oxygen binding
    Fairly prevalent in erythrocytes (4.5 mM);
     roughly equal to [Hb]
    Hb tetramer has one BPG binding site
    BPG effectively crosslinks the 2  chains
    It only fits in T (deoxy) form!

11/04/2008   Biochemistry: Regulation               p. 38 of 44
 BPG and physiology

    pO2 is too high (40 Torr) for efficient
     release of O2 in many cells in absence of
     BPG
    With BPG around, T-state is stabilized
     enough to facilitate O2 release
    Big animals (e.g. sheep) have lower O2
     affinity but their Hb is less influenced by
     BPG
11/04/2008   Biochemistry: Regulation    p. 39 of 44
 Fetal hemoglobin

    Higher oxygen affinity because the type
     of hemoglobin found there has a lower
     affinity for BPG
    Fetal Hb is 22;
      doesn’t bind BPG as much as .
    That helps ensure that plenty of O2 gets
     from mother to fetus across the placenta
11/04/2008   Biochemistry: Regulation   p. 40 of 44
 Sickle-cell anemia
   Genetic disorder: Hb residue 6 mutated from
    glu to val. This variant is called HbS.
   Results in intermolecular interaction between
    neighboring Hb tetramers that can cause
    chainlike polymerization
   Polymerized hemoglobin will partially fall out of
    solution and tug on the erythrocyte structure,
    resulting in misshapen (sickle-shaped) cells
   Oxygen affinity is lower because of insolubility



11/04/2008    Biochemistry: Regulation         p. 41 of 44
                                                  QuickTime™ an d a

    Why has this                         TIFF (Uncompressed) decompressor
                                            are need ed to see this p icture .



    mutation survived?
   Homozygotes don’t generally
    survive to produce progeny;                 Deoxy HbS
                                                    2.05 Å
    but heterozygotes do
                                                 PDB 2HBS
   Heterozygotes do have modestly reduced
    oxygen-carrying capacity in their blood because
    some erythrocytes are sickled
   BUT heterozygotes are somewhat resistant to
    malaria, so the gene survives in tropical places
    where malaria is a severe problem


11/04/2008    Biochemistry: Regulation                    p. 42 of 44
 How is sickling related
 to malaria?                                          QuickTime™ a nd a
                                             TIFF (Uncompressed) decompressor
                                                are need ed to see this picture.

   Malaria parasite (Plasmondium spp.)
    infects erythrocytes
   They’re unable to infect sickled cells
   So a partially affected cell might
    survive the infection better than a
    non-sickled cell
                                                   Plasmodium
   Still some argument about all of this          falciparum
   Note that most tropical environments           from A.Dove
    have plenty of oxygen around (not a            (2001) Nature
    lot of malaria at 2000 meters                  Medicine
    elevation)                                     7:389
11/04/2008   Biochemistry: Regulation              p. 43 of 44
 Other hemoglobin mutants
    Because it’s easy to get human blood,
     dozens of hemoglobin mutants have
     been characterized
    Many are asymptomatic
    Some have moderate to severe effects
     on oxygen carrying capacity or
     erythrocyte physiology

11/04/2008   Biochemistry: Regulation   p. 44 of 44

				
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