Chapter 15 Enzyme Regulation

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Chapter 15Enzyme Regulation
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Outline

• What factors influence enzymatic activity?
• What are the general features of allosteric
  regulation?
• Can allosteric regulation be explained by
  conformational changes in proteins?
• What kinds of covalent modification regulate the
  activity of enzymes?
• Is the activity of some enzymes controlled by
  both allosteric regulation and covalent
  modification?
• Special focus is there an example in nature
  that exemplifies the relationship between
  quaternary structure and the emergence of
  allosteric properties? Hemoglobin and Myoglobin
  paradigms of protein structure and function

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15.1 What Factors Influence Enzymatic Activity?

• The availability of substrates and cofactors
  usually determines how fast the reaction goes
• As product accumulates, the apparent rate of the
  enzymatic reaction will decrease
• Genetic regulation of enzyme synthesis and decay
  determines the amount of enzyme present at any
  moment
• Enzyme activity can be regulated allosterically
• Enzyme activity can be regulated through covalent
  modification
• Zymogens, isozymes, and modulator proteins may
  play a role

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15.1 What Factors Influence Enzymatic Activity?
Enzyme regulation by reversible covalent
modification.
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15.1 What Factors Influence Enzymatic Activity?
Zymogens are inactive precursors of enzymes.
Typically, proteolytic cleavage produces the
active enzyme.
Proinsulin is an 86-residue precursor to insulin
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The proteolytic activation of chymotrypsinogen
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Isozymes Are Enzymes With Slightly Different
Subunits
The isozymes of lactate dehydrogenase (LDH).
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15.2 What Are the General Features of
Allosteric Regulation?

• Action at "another site"
• Enzymes situated at key steps in metabolic
  pathways are modulated by allosteric effectors
• These effectors are usually produced elsewhere in
  the pathway
• Effectors may be feed-forward activators or
  feedback inhibitors
• Kinetics are sigmoid ("S-shaped")

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15.2 What Are the General Features of
Allosteric Regulation?
Sigmoid v versus S plot. The dotted line
represents the hyperbolic plot characteristic of
normal MichaelisMenten kinetics.
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15.3 Can Allosteric Regulation Be Explained by
Conformational Changes in Proteins?

• Monod, Wyman, Changeux (MWC) Model
• - Allosteric proteins can exist in two states R
  (relaxed) and T (taut)
• - In this model, all the subunits of an oligomer
  must be in the same state
• - T state predominates in the absence of
  substrate S
• - S binds much tighter to R than to T

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The Symmetry Model for Allosteric Regulation is
Based on Two Conformational States for a Protein
Allosteric effects A and I binding to R and T,
respectively.
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The Symmetry Model for Allosteric Regulation is
Based on Two Conformational States for a Protein
Allosteric effects A and I binding to R and T,
respectively.
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The Symmetry Model for Allosteric Regulation is
Based on Two Conformational States for a Protein
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More about the MWC model

• Cooperativity is achieved because S binding
  increases the population of R, which increases
  the sites available to S
• Ligands such as S are positive homotropic
  effectors
• Molecules that influence the binding of something
  other than themselves are heterotropic effectors

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The Sequential Model for Allosteric Regulation is
Based on Ligand-Induced Conformation Changes

• An alternative model proposed by Koshland,
  Nemethy, and Filmer (the KNF model) relies on the
  idea that ligand binding triggers a conformation
  change in a protein
• If the protein is oligomeric, ligand-induced
  conformation changes in one subunit may lead to
  conformation changes in adjacent subunits
• The KNF model explains how ligand-induced
  conformation changes could cause subunits to
  adopt conformations with little affinity for the
  ligand i.e., negative cooperativity
• The KNF model is termed the sequential model

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The Sequential Model for Allosteric Regulation is
Based on Ligand-Induced Conformation Changes
The Koshland-Nemethy-Filmer sequential model for
allosteric behavior. (a) S binding can, by
induced fit, cause a conformational change in the
subunit to which it binds. (b) If subunit
interactions are tightly coupled, binding of S to
one subunit may cause the other subunit to assume
a conformation having a greater or lesser
affinity for S. That is, the ligand-induced
conformational change in one subunit can affect
the adjoining subunit.
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The Sequential Model for Allosteric Regulation is
Based on Ligand-Induced Conformation Changes
The Koshland-Nemethy-Filmer model. Theoretical
curves for the binding of a ligand to a protein
having four identical subunits, each with one
binding site for the ligand.
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The notable difference between MWC and KNF models

• In the MWC model, the different conformations
  have different affinities for the various
  ligands, and the concept of ligand-induced
  conformational changes is ignored
• In contrast, the KNF model is based on
  ligand-induced conformational changes

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15.4 What Kinds of Covalent Modification Regulate
the Activity of Enzymes?

• Enzyme activity can be regulated through
  reversible phosphorylation
• This is the most prominent form of covalent
  modification in cellular regulation
• Phosphorylation is accomplished by protein
  kinases
• Each protein kinase targets specific proteins for
  phosphorylation
• Phosphoprotein phosphatases catalyze the reverse
  reaction removing phosphoryl groups from
  proteins
• Kinases and phosphatases themselves are targets
  of regulation

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15.4 What Kinds of Covalent Modification Regulate
the Activity of Enzymes?

• Protein kinases phosphorylate Ser, Thr, and Tyr
  residues in target proteins
• Kinases typically recognize specific amino acid
  sequences in their targets
• In spite of this specificity, all kinases share a
  common catalytic mechanism based on a conserved
  core kinase domain of about 260 residues (see
  Figure 15.9)
• Kinases are often regulated by intrasteric
  control, in which a regulatory subunit (or
  domain) has a pseudosubstrate sequence that
  mimics the target sequence, minus the
  phosphorylatable residue

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15.4 What Kinds of Covalent Modification Regulate
the Activity of Enzymes?
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Cyclic AMP-dependent protein kinase is composed
of catalytic and regulatory subunits
cyclic AMP-dependent protein kinase (also known
as protein kinase A (PKA) is a 150- to 170-kD
R2C2 tetramer in mammalian cells. The two R
(regulatory) subunits bind cAMP cAMP binding
releases the R subunits from the C (catalytic)
subunits. C subunits are enzymatically active as
monomers.
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Phosphorylation is Not the Only Form of Covalent
Modification that Regulates Protein Function

• Several hundred different chemical modifications
  of proteins have been discovered
• Only a few of these are used to achieve metabolic
  regulation through reversible conversion of an
  enzyme between active and inactive forms
• A few are summarized in Table 15.3
• Three of the modifications in Table 15.3 require
  nucleoside triphosphates (ATP, UTP) that are
  related to cellular energy status

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Phosphorylation is Not the Only Form of Covalent
Modification that Regulates Protein Function
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Acetylation in Enzyme Regulation

• Acetylation is a prominent modification for the
  regulation of metabolic enzymes
• Acetylation of an e-NH3 group on a Lys residue
  changes it from a positively charged amino group
  to a neutral amide
• This change may have consequences for protein
  structure and thus function
• The acetylating enzyme is termed an
  acetyl-CoA-dependent lysine acetyltransferase or
  KAT
• More than 30 KATs are known in mammals
• Deacetylation by KDACs (lysine deacetylases)
  reverse the effects of acetylation

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Acetylation in Enzyme Regulation

• Proteomics studies show that acetylation of
  metabolic enzymes is an important mechanism for
  regulating the flow of metabolic substrates
  (carbohydrates and fats, for example) through the
  central metabolic pathways
• Acetylation activates some enzymes and inhibits
  others
• Cellular levels of major metabolic fuels such as
  glucose, fatty acids, and amino acids influence
  the degree of acetylation
• The KDACs include sirtuins, a class of
  NAD-dependent protein deacetylating enzymes
• Sirtuins are implicated in energy metabolism and
  longevity

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15.5 Are Some Enzymes Controlled by Both
Allosteric Regulation and Covalent Modification?

• Glycogen phosphorylase (GP) is an example of the
  many enzymes that are regulated both by
  allosteric controls and by covalent modification
• GP cleaves glucose units from nonreducing ends of
  glycogen
• This converts glycogen into readily usable fuel
  in the form of glucose-1-phosphate
• This is a phosphorolysis reaction
• Muscle GP is a dimer of identical subunits, each
  with PLP covalently linked
• There is an allosteric effector site at the
  subunit interface

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Glycogen Phosphorylase is Controlled by Both
Allosteric Regulation and Covalent Modification
The mechanism of covalent modification and
allosteric regulation of glycogen phosphorylase.
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Glycogen phosphoryase is activated by a cascade
of reactions
The hormone-activated enzymatic cascade that
leads to activation of glycogen phosphorylase.
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Hemoglobin

• A classic example of allostery
• Hemoglobin and myoglobin are oxygen- transport
  and oxygen-storage proteins, respectively
• Compare the oxygen-binding curves for hemoglobin
  and myoglobin
• Myoglobin is monomeric hemoglobin is tetrameric
• Mb 153 aa, 17,200 MW
• Hb two a chains of 141 residues, 2 ß chains of
  146 residues

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Figure 15.21 O2-binding curves for hemoglobin and
myoglobin
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The structure of myoglobin is similar to that of
an Hb monomer
The myoglobin and hemoglobin structures. Myoglobi
n is monomeric Hemoglobin is tetrameric
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Mb and Hb use heme to bind Fe2
Heme is formed when protoporphyrin IX binds Fe2
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Fe2 is coordinated by His F8

• Iron interacts with six ligands in Hb and Mb
• Four of these are the N atoms of the porphyrin
• A fifth ligand is donated by the imidazole side
  chain of amino acid residue His F8
• (This residue is on the sixth or F helix, and
  it is the 8th residue in the helix, thus the
  name.)
• When Mb or Hb bind oxygen, the O2 molecule adds
  to the heme iron as the sixth ligand
• The O2 molecule is tilted relative to a
  perpendicular to the heme plane

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Fe2 is coordinated by His F8
The six liganding positions of an iron atom in Hb
and Mb.
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Myoglobin Structure

• Mb is a monomeric heme protein
• Mb polypeptide "cradles" the heme group
• Fe in Mb is Fe2 - ferrous iron - the form that
  binds oxygen
• Oxidation of Fe yields 3 charge - ferric iron
• Mb with Fe3 is called metmyoglobin and does not
  bind oxygen

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O2 Binding Alters Mb Conformation

• In deoxymyoglobin, the ferrous ion actually lies
  0.055 nm above the plane of the heme
• When oxygen binds to Fe in heme of Mb, the heme
  Fe is drawn toward the plane of the porphyrin
  ring
• With oxygen bound, the Fe2 atom is only 0.026 nm
  above the plane
• For Mb, this small change has little consequence
• But a similar change in Hb initiates a series of
  conformational changes that are transmitted to
  adjacent subunits

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Hb Has an a2ß2 Tetrameric Structure
An aß dimer of Hb, with packing contacts
indicated in blue. The sliding contacts made
with the other dimer are shown in yellow.
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Cooperative Binding of Oxygen Influences
Hemoglobin Function

• Mb, an oxygen-storage protein, has a greater
  affinity for oxygen at all oxygen pressures
• Hb is different it must bind oxygen in lungs
  and release it in capillaries
• Hb becomes saturated with O2 in the lungs, where
  the partial pressure of O2 is about 100 torr
• In capillaries, pO2 is about 40 torr, and oxygen
  is released from Hb
• The binding of O2 to Hb is cooperative binding
  of oxygen to the first subunit makes binding to
  the other subunits more favorable

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O2-Binding Curves of Mb and Hb
The oxygen binding curve of Mb resembles an
enzymesubstrate saturation curve.
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An Alternative O2-Binding Curve for Hb
Oxygen saturation curve for Hb in the form of Y
versus pO2 assuming n4 and P50 26 torr. Y is
the fractional saturation of Hb
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An Alternative O2-Binding Curve for Hb
A comparison of the experimentally observed O2
curve for Hb yielding a value for n of 2.8, the
hypothetical curve if n4, and the curve if n1
(non-interacting O2-binding sites).
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The Conformation Change

• The secret of Mb and Hb
• Oxygen binding changes the Mb conformation
• Without oxygen bound, Fe2 is out of heme plane
• Oxygen binding pulls the Fe2 into the heme plane
  
• Fe2 pulls its His F8 ligand along with it
• The F helix moves when oxygen binds
• Total movement of Fe2 is 0.029 nm i.e., 0.29 Å
  
• This change means little to Mb, but lots to Hb!

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Oxygen Binding by Hb Induces a Quaternary
Structure Change

• When deoxy-Hb crystals are exposed to oxygen,
  they shatter. Evidence of a large-scale
  structural change
• One alpha-beta pair moves relative to the other
  by 15 degrees upon oxygen binding
• This massive change is induced by movement of Fe
  by 0.039 nm when oxygen binds

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Oxygen binding to Hb results in a 15 rotation of
one aß pair relative to the other
Subunit motion in hemoglobin when the molecule
goes from the (a) deoxy form to the (b) oxy form.
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Fe2 Movement by Less Than 0.04 nm Induces the
Conformation Change in Hb

• In deoxy-Hb, the iron atom lies out of the heme
  plane by about 0.06 nm
• Upon O2 binding, the Fe2 atom moves about 0.039
  nm closer to the plane of the heme
• It is as if the O2 is drawing the heme iron into
  the plane
• This may seem like a trivial change, but its
  biological consequences are far-reaching
• As Fe2 moves, it drags His F8 and the F helix
  with it
• This change is transmitted to the subunit
  interfaces, where conformation changes lead to
  the rupture of salt bridges

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Fe2 Movement by Less Than 0.04 nm Induces the
Conformation Change in Hb
Changes in the position of the heme iron atom
upon oxygenation lead to conformational changes
in the hemoglobin molecule.
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The Physiological Significance of the HbO2
Interaction

• Hb must be able to bind oxygen in the lungs
• Hb must be able to release oxygen in capillaries
• If Hb behaved like Mb, very little oxygen would
  be released in capillaries
• The sigmoid, cooperative oxygen-binding curve of
  Hb makes its physiological actions possible!

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H Promotes Dissociation of Oxygen from Hemoglobin

• Binding of O2 to Hb is affected by several
  agents, including H, CO2, and chloride ions
• The effect of H is particularly important
• Deoxy-Hb has a higher affinity for H than oxy-Hb
• Thus, as pH decreases, dissociation of O2 from
  hemoglobin is enhanced
• Ignoring the stoichiometry of O2 and H, we can
  write
• HbO2 H ? HbH CO2

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H Promotes Dissociation of Oxygen from Hemoglobin
The oxygen saturation curves for myoglobin and
for hemoglobin at five different pH values 7.6,
7.4,7.2, 7.0, 6.8.
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The Antagonism of O2 Binding by H is Termed the
Bohr Effect

• The effect of H on O2 binding was discovered by
  Christian Bohr (the father of Neils Bohr, the
  atomic physicist)
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance

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CO2 Also Promotes the Dissociation of O2 from
Hemoglobin

• Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues and extremities leads
  to proton production
• CO2 H2O ? H HCO3
• These protons are taken up by Hb as oxygen
  dissociates
• The reverse occurs in the lungs

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CO2 Also Promotes the Dissociation of O2 from
Hemoglobin
Oxygen binding curves of blood and of hemoglobin
in the absence and presence of CO2 and BPG.
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Summary of the Physiological Effects of H and
CO2 on O2 Binding by Hemoglobin

• At the tissue-capillary interface, CO2 hydration
  and glycolysis produce extra H, promoting
  additional dissociation of O2 where it is needed
  most
• At the lung-artery interface, bicarbonate
  dehydration (required for CO2 exhalation)
  consumes extra H, promoting CO2 release and O2
  binding

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2,3-Bisphosphoglycerate

• An Allosteric Effector of Hemoglobin
• In the absence of 2,3-BPG, oxygen binding to Hb
  follows a rectangular hyperbola!
• The sigmoid binding curve is only observed in the
  presence of 2,3-BPG
• Since 2,3-BPG binds at a site distant from the Fe
  where oxygen binds, it is called an allosteric
  effector

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BPG Binding to Hb Has Important Physiological
Significance

• The "inside" story......
• Where does 2,3-BPG bind?
• "Inside"
• in the central cavity
• What is special about 2,3-BPG?
• Negative charges interact with 8 positive charges
  in the cavity 2 Lys, 4 His, 2 N-termini
• Fetal Hb - lower affinity for 2,3-BPG, higher
  affinity for oxygen, so it can get oxygen from
  mother

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Fetal Hemoglobin Has a Higher Affinity for O2
Because it has a Lower Affinity for BPG

• The fetus depends on its mother for O2, but its
  circulatory system is entirely independent
• Gas exchange takes place across the placenta
• Fetal Hb differs from adult Hb with ?-chains in
  place of ß-chains and thus a a2?2 structure
• As a result, fetal Hb has a higher affinity for
  O2
• Why does fetal Hb bind O2 more tightly?
• Fetal ?-chains have Ser instead of His at
  position 143 and thus lack two of the positive
  charges in the BPG-binding cavity
• BPG binds less tightly and Hb F thus looks more
  like Mb in its O2 binding behavior

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Fetal Hemoglobin Has a Higher Affinity for O2
Because it has a Lower Affinity for BPG
Comparison of the oxygen saturation curves of Hb
A and Hb F under similar conditions of pH and
BPG.
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Sickle-Cell Anemia is a Molecular Disease

• Sickle-cell anemia patients have
  abnormally-shaped red blood cells
• The erythrocytes are crescent-shaped instead of
  disc-shaped
• The sickle cells pass less freely through the
  capillaries, impairing circulation and causing
  tissue damage
• A single amino acid substitution in the ß-chains
  of Hb causes sickle-cell anemia
• Glu at position 6 of the ß-chains is replaced by
  Val
• As a result, Hb S molecules aggregate into long,
  chainlike polymeric structures

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Sickle-Cell Anemia is a Molecular Disease
The polymerization of Hb S molecules arises
because Val replaces His on the surface of
ß-chains. The block extending from Hb S below
represents the Val side chains. These can insert
into hydrophobic pockets in neighboring Hb S
molecules.
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Sickle-Cell Anemia is a Molecular Disease
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Sickle-Cell Anemia is a Molecular Disease
Structure of the polymerized Hb S filament. Val
at position 6 of the ß-chains is shown in blue.
Hemes are red.