Transcript Chapter 15 - Richsingiser.com

Reginald H. Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett
Chapter 15
Enzyme Regulation
Reginald Garrett & Charles Grisham • University of Virginia
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
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
15.1 – What Factors Influence Enzymatic
Activity?
Enzyme regulation by reversible covalent modification.
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
The proteolytic activation of chymotrypsinogen
Isozymes Are Enzymes With Slightly
Different Subunits
The isozymes of
lactate dehydrogenase
(LDH).
15.2 – What Are the General Features of
Allosteric Regulation?
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•
•
•
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")
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 Michaelis=Menten kinetics.
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
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.
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.
The Symmetry Model for Allosteric Regulation is
Based on Two Conformational States for a Protein
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
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
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.
The Sequential Model for Allosteric Regulation is
Based on Ligand-Induced Conformation Changes
The KoshlandNemethy-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.
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 ligandinduced conformational changes
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
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
15.4 What Kinds of Covalent Modification
Regulate the Activity of Enzymes?
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.
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
Phosphorylation is Not the Only Form of Covalent
Modification that Regulates Protein Function
Acetylation in Enzyme Regulation
• Acetylation is a prominent modification for the
regulation of metabolic enzymes
• Acetylation of an ε-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-CoAdependent lysine acetyltransferase or KAT
• More than 30 KATs are known in mammals
• Deacetylation by KDACs (lysine deacetylases)
reverse the effects of acetylation
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
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
Glycogen Phosphorylase is Controlled by Both
Allosteric Regulation and Covalent Modification
The mechanism of covalent
modification and allosteric
regulation of glycogen
phosphorylase.
Glycogen phosphoryase is activated by a
cascade of reactions
The hormone-activated enzymatic cascade that leads to
activation of glycogen phosphorylase.
Hemoglobin
A classic example of allostery
• Hemoglobin and myoglobin are oxygentransport 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 α chains of 141 residues, 2 β chains of
146 residues
Figure 15.21 O2-binding curves for hemoglobin and
myoglobin
The structure of myoglobin is similar to that
of an Hb monomer
The myoglobin and hemoglobin
structures.
Myoglobin is monomeric
Hemoglobin is tetrameric
Mb and Hb use heme to bind Fe2+
Heme is formed when protoporphyrin IX binds Fe2+
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
Fe2+ is coordinated by His F8
The six liganding positions
of an iron atom in Hb and
Mb.
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
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
Hb Has an α2β2 Tetrameric Structure
An αβ dimer of Hb,
with packing contacts
indicated in blue.
The sliding contacts
made with the other
dimer are shown in
yellow.
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
O2-Binding Curves of Mb and Hb
The oxygen binding curve of
Mb resembles an
enzyme:substrate saturation
curve.
An Alternative O2-Binding Curve for Hb
Oxygen saturation
curve for Hb in
the form of Y
versus pO2
assuming n=4
and P50 =26 torr.
Y is the fractional
saturation of Hb:
[ pO2 ]
Y=
4
[ pO2 ] + K
4
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 n=4, and
the curve if n=1
(non-interacting
O2-binding sites).
The Conformation Change
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•
•
•
•
•
•
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!
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
Oxygen binding to Hb results in a 15° rotation of
one αβ pair relative to the other
Subunit motion in hemoglobin when the molecule goes from
the (a) deoxy form to the (b) oxy form.
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
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.
The Physiological Significance of the Hb:O2
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!
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
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.
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
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
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.
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
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
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
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 γ-chains in
place of β-chains – and thus a α2γ2 structure
• As a result, fetal Hb has a higher affinity for O2
• Why does fetal Hb bind O2 more tightly?
• Fetal γ-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
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].
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 β-chains of Hb
causes sickle-cell anemia
• Glu at position 6 of the β-chains is replaced by Val
• As a result, Hb S molecules aggregate into long,
chainlike polymeric structures
Sickle-Cell Anemia is a Molecular Disease
The polymerization of Hb S molecules arises because Val replaces His on the surface
of β-chains. The “block” extending from Hb S below represents the Val side chains.
These can insert into hydrophobic pockets in neighboring Hb S molecules.
Sickle-Cell Anemia is a Molecular Disease
Sickle-Cell Anemia is a Molecular Disease
Structure of the polymerized Hb S filament. Val at position 6
of the β-chains is shown in blue. Hemes are red.