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 Allosteric Regulation Be Explained by
   Conformational Changes in Proteins?
4. What Kinds of Covalent Modification Regulate the
   Activity of Enzymes?
5. 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 protein degradation
  (enzyme level)(chapter 2931)
• Enzyme activity can be regulated allosterically
• Enzyme activity can be regulated by covalent
  modification
• Zymogens, isozymes and modulator proteins may
  play a role

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Figure 15.1 Enzyme regulation by reversible
covalent modification.
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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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formation of a blood clot.
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Isozymes
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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
• F, 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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• 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 (more than 1 polypeptide)
• The regulatory effects exerted on the enzymes
  activity are achieved by comformational changes
  occurring in the protein when effector
  metabolites bind

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15.3 Can Allosteric Regulation Be Explained by
Conformational Changes in Proteins?

• Symmetry model Two conformational states
• Monod, Wyman, Changeux (MWC) Model allosteric
  proteins can exist in two states R (relaxed) and
  T (taut) R0 ? T0
• In this model, all the subunits in an oligomer
  must be in the same state (R or T)
• T-state predominates in the absence of substrate
  S
• The substrate and activators bind only to the
  R-state and inhibitor bind only to T-state

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Figure 15.7 Heterotropic allosteric effects A
and I binding to R and T, respectively.
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• Although the relative R0 concentration is
  small, S will bind only to R0, forming R1
• S binds much tighter to R than to T
• 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
• K0.5 (Km) the concentration of ligand giving
  half-maximal response
• Ligands such as S are positive homotropic
  effectors

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• Molecules that influence the binding of something
  other than themselves are heterotropic effectors
• Positive heterotropic effectors or allosteric
  avtivators (T0 ? R0) cause a decline in the K0.5
  for S
• negative heterotropic effectors or allosteric
  inhibitors (R0 ? T0) raise K0.5 for S
• The MWC model assumes an equilibrium between
  conformational states, but ligand binding does
  not alter the conformation of the protein

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• The sequential 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
• Ligand-induced conformation changes could cause
  subunits to shift from a low-affinity state to a
  high-affinity state
• The sequential model means subunits undergo
  sequential changes in conformation due to ligand
  binding

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Figure 15.8 The KNF sequential model for
allosteric behavior.
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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
• Protein kinases and phosphatases work in opposing
  directions

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Figure 15.1 Enzyme regulation by reversible
covalent modification.
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• Protein kinases
• phosphorylate Ser, Thr, and Tyr residues in
  target proteins (Table 15.2)

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• Phosphorylation introduces a bulky group bearing
  two negative charges, causing conformational
  changes that alter the target proteins function
• 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)
• Protein kinases are classified as Ser/Thr and/or
  Tyr specific
• Kinases are often regulated by intrasteric
  control (see Figure 15.10)

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This complex also includes ATP (red) and two Mn2
ions (yellow) bound at the active site. Figure
15.9 Protein kinase A is shown complexes with a
pseudosubstrate peptide (orange).
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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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Figure 25.16 Covalent modification of GS
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Phosphorylation
Adenylylation
ADP-ribosylation
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15.5 Is the Activity of Some Enzymes Controlled
by Both Allosteric Regulation and Covalent
Modification?

• Glycogen phosphorylase (GP)
• Regulated both by allosteric regulation and by
  covalent modification
• Catalyzes the release of glucose units from
  glycogen
• A phosphorolysis reaction (Figure 15.11) produces
  glucose-1-phosphate which is converted to
  glucose-6-P
• In muscle, glucose-6-P proceeds into glycolysis,
  providing needed energy for muscle contraction
• In the liver, hyrdolysis of glucose-6-P yield
  glucose, which is exported to other tissues

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Figure 15.11 The glycogen phosphorylase
reaction.
Figure 15.12 The phosphoglucomutase reaction.
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• GP is a homodimer
• Muscle glycogen phosphorylase is a dimer of two
  identical subunits (842 residues)
• Each subunit contains an active site
• A pyridoxal phosphate cofactor covalently linked
  (Lys-680)
• 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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GP Activity is Regulated Allosterically

• Cooperativity in substrate binding (15.14a)
• Inorganic phosphate (Pi) is a positive homotropic
  effector
• ATP is a feedback inhibitor, and a allosteric
  inhibitor
• Glucose-6-P is a negative heterotropic effector
  (i.e., an allosteric inhibitor)
• AMP is a positive heterotropic effector (i.e., an
  allosteric activator)

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Figure 15.14 v versus S curves for glycogen
phosphorylase. The response to the concentration
of the substrate phosphate (Pi). ATP is a
feedback inhibitor. AMP is a positive effector.
It binds at the same site as ATP.
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• AMP and ATP bind to the same site
• AMP promotes the conversion to the active state
• ATP, glucose-6-P, and caffeine favor conversion
  to the inactive state
• GP conforms to the MWC model of allosteric
  transition (T and R conversion)
• The active form of the enzyme is designated the R
  state
• The inactive form of the enzyme is denoted the T
  state
• Allosteric controls can be overridden by covalent
  modiffication of GP

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Figure 15.15The 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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Covalent Modification of GP Trumps Allosteric
Regulation

• In 1956, Edwin Krebs and Edmond Fischer showed
  that a converting enzyme could convert
  phosphorylase b (inactive) to phosphorylase a
  (active)
• Three years later, Krebs and Fischer show that
  this conversion involves covalent phosphorylation
  (Figure 15.15)

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Figure 15.16 The major conformational change
that occurs in the N-terminal residues upon
phosphorylation of Ser14. Ser14 is shown in red.
N-terminal conformation of unphosphorylated
enzyme (phosphorylase b) cyan. N-terminal
conformation of phosphorylated enzyme
(phosphorylase a) yellow.
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Enzyme cascades regulate GP Covalent Modification

• This phosphorylation of GP is mediated by an
  enzyme cascade (Figure 15.17)
• Leads to hormonal stimulation of adenylyl cyclase
  that converts ATP to cAMP
• Cyclic AMP is the intracellular agent of
  extracellular hormones is known as a second
  messenger (chap 32)

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Figure 15.17 The hormone-activated enzymatic
cascade that leads to activation of glycogen
phosphorylase.
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• The hormonal stimulation of adenylyl cyclase is
  effected by a transmembrane signal pathway
• Hormone binding stimulates a GTP-binding protein
  (G protein Gabg)
• G? has GTPase activity and binds GDP or GTP
• Gabg complex has GDP at the nucleotide site
• When stimulated, GDP dissociates and GTP binds to
  G?
• G? dissociates from Gbg and associates with
  adenylyl cyclase
• Binding of G? stimulates adenylyl cyclase to make
  cAMP
• GTPase activity of G? hydrolyzes GTP to GDP,
  leading to dissociation of G? from adenylyl
  cyclase and reassociation with Gbg to form Gabg
• cAMP is an essential activator of cAMP-dependent
  protein kinase (PKA)

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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 residues

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Figure 15.20 O2-binding curves for hemoglobin and
myoglobin.  
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(No Transcript)
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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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Fetal hemoglobin has a higher affinity for O2
because it has a lower affinity for BPG
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Sickle cell anemia