Enzyme Regulation

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Chapter 15

• Enzyme Regulation
• Biochemistry
• by
• Reginald Garrett and Charles Grisham

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Essential Question

1. What are the properties of regulatory enzymes?
2. How do regulatory enzymes sense the momentary
   needs of cells?
3. What molecular mechanisms are used to regulate
   enzyme activity?

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Outline of Chapter 15

1. What Factors Influence Enzymatic Activity?
2. What Are the General Features of Allosteric
   Regulation?
3. Can a Simple Equilibrium Model Explain Allosteric
   Kinetics?
4. Is the Activity of Some Enzymes Controlled by
   Both Allosteric Regulation and Covalent
   Modification?

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15.1 What Factors Influence Enzymatic Activity?

• The activity displayed by enzymes is affected by
  a variety of factors, some of which are essential
  to the harmony of metabolism
• Two of the more obvious ways to regulate the
  amount of activity are
• To increase or decrease the number of enzyme
  molecule (enzyme level)
• To increase or decrease the activity of each
  enzyme molecule (enzyme activity)

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• A general overview of factors influencing enzyme
  activity includes the following considerations
• Rate depends on substrate availability
• Rate slows as product accumulates
• Genetic controls (transcription regulation) -
  induction and repression (enzyme level)
• Allosteric effectors may be important
• Enzymes can be modified covalently
• Zymogens, isozymes and modulator proteins may
  play a role

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Figure 15.1Enzymes regulated by covalent
modification are called interconvertible enzymes.
The enzymes (protein kinase and protein
phosphatase, in the example shown here)
catalyzing the conversion of the interconvertible
enzyme between its two forms are called converter
enzymes. In this example, the free enzyme form is
catalytically active, whereas the
phosphoryl-enzyme form represents an inactive
state. The -OH on the interconvertible enzyme
represents an -OH group on a specific amino acid
side chain in the protein (for example, a
particular Ser residue) capable of accepting the
phosphoryl group.
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Phosphorylation
Adenylylation
ADP-ribosylation
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• A general overview of factors influencing enzyme
  activity includes the following considerations
• Rate depends on substrate availability
• Rate slows as product accumulates
• Genetic controls (transcription regulation) -
  induction and repression (enzyme level)
• Allosteric effectors may be important
• Enzymes can be modified covalently
• Zymogens, isozymes and modulator proteins may
  play a role

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Zymogens
Figure 15.2Proinsulin is an 86-residue precursor
to insulin (the sequence shown here is human
proinsulin). Proteolytic removal of residues 31
to 65 yields insulin. Residues 1 through 30 (the
B chain) remain linked to residues 66 through 87
(the A chain) by a pair of interchain disulfide
bridges.
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Figure 15.3 The proteolytic activation of
chymotrypsinogen.
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Figure 15.4The cascade of activation steps
leading to blood clotting. The intrinsic and
extrinsic pathways converge at Factor X, and the
final common pathway involves the activation of
thrombin and its conversion of fibrinogen into
fibrin, which aggregates into ordered filamentous
arrays that become cross-linked to form the clot.
Serine protease Kallikrein VIIa IXa Xa XIa XIIa T
hronbin
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Rich in negative charge
formation of a blood clot.
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Isozymes
Figure 18.30(a) Pyruvate reduction to ethanol in
yeast provides a means for regenerating NAD
consumed in the glyceraldehyde-3-P dehydrogenase
reaction. (b) In oxygen-depleted muscle, NAD is
regenerated in the lactate dehydrogenase
reaction.
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Figure 15.5 The isozymes of lactate
dehydrogenase (LDH). Active muscle tissue becomes
anaerobic and produces pyruvate from glucose via
glycolysis (Chapter 18). It needs LDH to
regenerate NAD from NADH so glycolysis can
continue. The lactate produced is released into
the blood. The muscle LDH isozyme (A4) works best
in the NAD-regenerating direction. Heart tissue
is aerobic and uses lactate as a fuel, converting
it to pyruvate via LDH and using the pyruvate to
fuel the citric acid cycle to obtain energy. The
heart LDH isozyme (B4) is inhibited by excess
pyruvate so the fuel wont be wasted.
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• Modulator proteins are another way that cells
  mediate metabolic activity
• cAMP-dependent protein kinase
• Phosphoprotein phosphatase inhibitor-I

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Figure 15.6Cyclic AMP- dependent protein kinase
(also known as PKA) is a 150- to 170-kD R2C2
tetramer in mammalian cells. The two R
(regulatory) subunits bind cAMP (KD 3 x 10-8
M) cAMP binding releases the R subunits from the
C (catalytic) subunits. C subunits are
enzymatically active as monomers.
Cyclic AMP-dependent protein kinase is shown
complexed with a pseudosubstrate peptide (red). 
This complex also includes ATP (yellow) and two
Mn2 ions (violet) bound at the active site.
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Figure 15.1Enzymes regulated by covalent
modification are called interconvertible enzymes.
The enzymes (protein kinase and protein
phosphatase, in the example shown here)
catalyzing the conversion of the interconvertible
enzyme between its two forms are called converter
enzymes. In this example, the free enzyme form is
catalytically active, whereas the
phosphoryl-enzyme form represents an inactive
state. The -OH on the interconvertible enzyme
represents an -OH group on a specific amino acid
side chain in the protein (for example, a
particular Ser residue) capable of accepting the
phosphoryl group.
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15.2 What Are the General Features of
Allosteric Regulation?

• Action at "another site"
• Allosteric regulation acts to modulate enzymes
  situated at key steps in metabolic pathways
• A ? B ? C ? D ? E ? F
• E, the essential end product, inhibits enzyme 1,
  the first step in the pathway
• This phenomenon is called feedback inhibition or
  feedback regulation

Enz 1
Enz 2
Enz 3
Enz 4
Enz 5
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• Regulatory enzymes have certain exceptional
  properties
• Their kinetics do not obey the Michaelis-Menten
  equation
• Their v versus S plots yield sigmoid- or
  S-shaped curve
• A second-order (or higher) relationship between v
  and S
• Substrate binding is cooperative

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Figure 15.7  Sigmoid v versus S plot. The
dotted line represents the hyperbolic plot
characteristic of normal Michaelis - Menten-type
enzyme kinetics.
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• Regulatory enzymes have certain exceptional
  properties
• Their kinetics do not obey the Michaelis-Menten
  equation
• Inhibition of a regulatory enzyme by a feedback
  inhibitor does not conform to any normal
  inhibition pattern- Allosteric inhibition
• Some effector molecules exert negative effects on
  enzyme activity, other effectors show
  stimulatory, or positive, influences on activity
• Oligomeric organization
• The regulatory effects exerted on the enzymes
  activity are achieved by comformational changes
  occurring in the proetin when effector
  metabolites bind

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15.3 Can a Simple Equilibrium Model Explain
Allosteric Kinetics?

• 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 (R or T)
• T state predominates in the absence of substrate
  S
• R0 ? T0
• L T0 / R0
• L is assume to be large (T ?? R)
• S binds much tighter to R than to T

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Figure 15.8 Monod - Wyman - Changeux (MWC) model
for allosteric transitions. Consider a dimeric
protein that can exist in either of two
conformational states, R or T. Each subunit in
the dimer has a binding site for substrate S and
an allosteric effector site, F. The promoters are
symmetrically related to one another in the
protein, and symmetry is conserved regardless of
the conformational state of the protein. The
different states of the protein, with or without
bound ligand, are linked to one another through
the various equilibria. Thus, the relative
population of protein molecules in the R or T
state is a function of these equilibria and the
concentration of the various ligands, substrate
(S), and effectors (which bind at FR or FT). As
S is increased, the T/R equilibrium shifts in
favor of an increased proportion of R-conformers
in the total population (that is, more protein
molecules in the R conformational state).
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• Although the relative R0 concentration is
  small, S will bind only to R0, forming R1
• S-binding drives the conformation transition,
• T0 ? R0
• 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

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Figure 15.9  The Monod - Wyman - Changeux model.
Graphs of allosteric effects for a tetramer (n
4) in terms of Y, the saturation function, versus
S. Y is defined as ligand-binding sites that
are occupied by ligand/ total ligand-binding
sites. (a) A plot of Y as a function of S, at
various L values. (b) Y as a function of S, at
different c, where c KR/KT. (When c 0, KT is
infinite.) (Adapted from Monod, J., Wyman, J.,
and Changeux, J.-P., 1965. On the nature of
allosteric transitions A plausible model.
Journal of Molecular Biology 1292.)
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• Molecules that influence the binding of something
  other than themselves are heterotropic effectors
• Positive heterotropic effectors or allosteric
  avtivators
• negative heterotropic effectors or allosteric
  inhibitors

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Figure 15.10 Heterotropic allosteric effects A
and I binding to R and T, respectively. The
linked equilibria lead to changes in the relative
amounts of R and T and, therefore, shifts in the
substrate saturation curve. This behavior,
depicted by the graph, defines an allosteric K
system. The parameters of such a system are (1)
S and A (or I) have different affinities for R
and T and (2) A (or I) modifies the apparent K0.5
for S by shifting the relative R versus T
population.
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• K system and V system are two different forms of
  the MWC model
• In K system
• The concentration of S giving half-maximal
  velocity, defined as K0.5, changes in response to
  effectors
• Vmax is constant
• In V system
• K0.5 is constant
• Vmax change
• V versus S plots are hyperbolic rather than
  S-shaped

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15.4 Is the Activity of Some Enzymes Controlled
by Both Allosteric Regulation and Covalent
Modification?

• Allosteric Regulation and Covalent Modification
• Glycogen phosphorylase cleaves glucose units from
  nonreducing ends of glycogen
• A phosphorolysis reaction
• Muscle glycogen phosphorylase 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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Figure 15.12 The glycogen phosphorylase
reaction.
Figure 15.13The phosphoglucomutase reaction.
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• Muscle glycogen phosphorylase is a dimer of two
  identical subunits (842 residues)
• Each subunit contains a pyridoxal phosphate
  cofactor covalently linked (Lys-680)
• An active site
• An allosteric effector site near the subunit
  interface
• A regulatory phosphorylation site (Ser-14)
• A glycogen binding site
• A tower helix (residues 262 to 278)

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Figure 15.14  (a) The structure of a glycogen
phosphorylase monomer, showing the locations of
the catalytic site, the PLP cofactor site, the
allosteric effector site, the glycogen storage
site, the tower helix (residues 262 through 278),
and the subunit interface. (b) Glycogen
phosphorylase dimer.
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Allosteric Regulation of GP

• Cooperativity in substrate binding (15.15a)
• Inorganic phosphate (Pi)is a positive homotropic
  effector
• ATP is a feedback inhibitor, and a negative
  heterotropic effector
• Glucose-6-P is a negative heterotropic effector
  (i.e., an inhibitor)
• AMP is a positive heterotrophic effector (i.e.,
  an activator)

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Figure 15.15v versus S curves for glycogen
phosphorylase. (a) The sigmoid response of
glycogen phosphorylase to the concentration of
the substrate phosphate (Pi) shows strong
positive cooperativity. (b) ATP is a feedback
inhibitor that affects the affinity of glycogen
phosphorylase for its substrates but does not
affect Vmax. (Glucose-6-P shows similar effects
on glycogen phosphorylase.) (c) AMP is a positive
heterotropic effector for glycogen phosphorylase.
It binds at the same site as ATP. AMP and ATP are
competitive. Like ATP, AMP affects the affinity
of glycogen phosphorylase for its substrates, but
does not affect Vmax.
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Figure 15.16 The mechanism of covalent
modification and allosteric regulation of
glycogen phosphorylase. The T states are blue and
the R states blue-green.
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Regulation of GP by Covalent Modification

• In 1956, Edwin Krebs and Edmond Fischer showed
  that a converting enzyme could convert
  phosphorylase b to phosphorylase a
• Three years later, Krebs and Fischer show that
  this conversion involves covalent phosphorylation
• This phosphorylation is mediated by an enzyme
  cascade (Figure 15.18)

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Figure 15.17In this diagram of the glycogen
phosphorylase dimer, the phosphorylation site
(Ser14) and the allosteric (AMP) site face the
viewer. Access to the catalytic site is from the
opposite side of the protein. The diagram shows
the major conformational change that occurs in
the N-terminal residues upon phosphorylation of
Ser14. The solid black line shows the
conformation of residues 10 to 23 in the b, or
unphosphorylated, form of glycogen phosphorylase.
The conformational change in the location of
residues 10 to 23 upon phosphorylation of Ser14
to give the a (phosphorylated) form of glycogen
phosphorylase is shown in yellow. Note that these
residues move from intrasubunit contacts into
intersubunit contacts at the subunit interface.
Sites on the two respective subunits are
denoted, with those of the upper subunit
designated by primes (). (Adapted from
Johnson, L. N., and Barford, D., 1993. The
effects of phosphorylation on the structure and
function of proteins. Annual Review of Biophysics
and Biomolecular Structure 22199-232.)
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Figure 15.18 The hormone-activated enzymatic
cascade that leads to activation of glycogen
phosphorylase.
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cAMP is a Second Messenger

• Cyclic AMP is the intracellular agent of
  extracellular hormones - thus a second
  messenger
• Hormone binding stimulates a GTP-binding protein
  (G protein), releasing G?(GTP)
• Binding of G?(GTP) stimulates adenylyl cyclase to
  make cAMP

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Figure 15.19 The adenylyl cyclase reaction
yields 3',5' -cyclic AMP and pyrophosphate. The
reaction is driven forward by subsequent
hydrolysis of pyrophosphate by the enzyme
inorganic pyrophosphatase.
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Figure 15.20Hormone (H) binding to its receptor
(R) creates a hormonereceptor complex (HR) that
catalyzes GDP-GTP exchange on the a -subunit of
the heterotrimer G protein (Gabg ), replacing GDP
with GTP. The Ga -subunit with GTP bound
dissociates from the bg -subunits and binds to
adenylyl cyclase (AC). AC becomes active upon
association with Ga GTP and catalyzes the
formation of cAMP from ATP. With time, the
intrinsic GTPase activity of the Ga -subunit
hydrolyzes the bound GTP, forming GDP this leads
to dissociation of Ga GDP from AC, reassociation
of Ga with the bg subunits, and cessation of AC
activity. AC and the hormone receptor H are
integral plasma membrane proteins Ga and Gbg are
membrane-anchored proteins.
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Hemoglobin

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

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Figure 15.21O2-binding curves for hemoglobin and
myoglobin.  
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Hemoglobin Function Hb must bind oxygen in lungs
and release it in capillaries

• Adjacent subunits' affinity for oxygen increases
• This is called positive cooperativity

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The Bohr Effect

• Competition between oxygen and H
• Discovered by Christian Bohr
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance
• See Figure 15.33

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Figure 15.33  The oxygen saturation curves for
myoglobin and for hemoglobin at five different pH
values 7.6, 7.4, 7.2, 7.0, and 6.8.
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Bohr Effect II

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

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Figure 15.34Oxygen-binding curves of blood and
of hemoglobin in the absence and presence of CO2
and BPG. From left to right stripped Hb, Hb
CO2, Hb BPG, Hb BPG CO2, and whole blood.
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