Transcript CHAPTER 6

Chapter 15
Enzyme Regulation
Biochemistry
by
Reginald Garrett and Charles Grisham
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?
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?
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
1. To increase or decrease the number of enzyme
molecule (enzyme level)
2. To increase or decrease the activity of each
enzyme molecule (enzyme activity)
A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Genetic controls (transcription regulation) induction and repression & protein degradation
(enzyme level)(chapter 29&31)
4. Enzyme activity can be regulated allosterically
5. Enzyme activity can be regulated by covalent
modification
6. Zymogens, isozymes and modulator proteins
may play a role
Figure 15.1 Enzyme regulation by reversible covalent
modification.
Zymogens
Figure 15.2
Proinsulin is an 86residue 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.
Figure 15.3 The proteolytic
activation of chymotrypsinogen.
Figure 15.4
The 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
Thronbin
formation of a blood clot.
Isozymes
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
•
Enz 1
Enz 2
Enz 3
Enz 4
Enz 5
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
Regulatory enzymes have certain exceptional
properties
1. Their kinetics do not obey the MichaelisMenten 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
• Regulatory enzymes have certain exceptional
properties
1. Their kinetics do not obey the Michaelis-Menten
equation
2. Inhibition of a regulatory enzyme by a feedback
inhibitor does not conform to any normal
inhibition pattern- Allosteric inhibition
3. Some effector molecules exert negative effects on
enzyme activity, other effectors show stimulatory,
or positive, influences on activity
4. Oligomeric organization (more than 1 polypeptide)
5. The regulatory effects exerted on the enzyme’s
activity are achieved by comformational changes
occurring in the protein when effector metabolites
bind
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 Rstate and inhibitor bind only to T-state
Figure 15.7
Heterotropic allosteric effects: A and I binding to R and T, respectively.
• 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 halfmaximal response
• Ligands such as S are positive homotropic
effectors
• 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
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 highaffinity state
• The sequential model means subunits undergo
sequential changes in conformation due to ligand
binding
Figure 15.8 The KNF
sequential model for
allosteric behavior.
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
Figure 15.1 Enzyme regulation by reversible covalent
modification.
Protein kinases:
• phosphorylate Ser, Thr, and Tyr residues in
target proteins (Table 15.2)
• Phosphorylation introduces a bulky group
bearing two negative charges, causing
conformational changes that alter the target
protein’s 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)
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).
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
Figure 25.16 Covalent modification of GS
Phosphorylation
Adenylylation
ADP-ribosylation
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
Figure 15.11 The
glycogen phosphorylase
reaction.
Figure 15.12 The
phosphoglucomutase
reaction.
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)
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)
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.
• 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
Figure 15.15
The mechanism of covalent
modification and allosteric
regulation of glycogen
phosphorylase. The T states
are blue and the R states
blue-green.
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)
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.
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)
Figure 15.17 The hormone-activated enzymatic cascade that leads to activation of
glycogen phosphorylase.
The hormonal stimulation of adenylyl cyclase
is effected by a transmembrane signal
pathway
– Hormone binding stimulates a GTP-binding
protein (G protein; Gabg)
– Ga has GTPase activity and binds GDP or GTP
– Gabg complex has GDP at the nucleotide site
– When stimulated, GDP dissociates and GTP
binds to Ga
– Ga dissociates from Gbg and associates with
adenylyl cyclase
– Binding of Ga stimulates adenylyl cyclase to
make cAMP
– GTPase activity of Ga hydrolyzes GTP to GDP,
leading to dissociation of Ga from adenylyl
cyclase and reassociation with Gbg to form Gabg
– cAMP is an essential activator of cAMPdependent protein kinase (PKA)
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
Figure 15.20 O2-binding curves for hemoglobin and myoglobin.
Hemoglobin Function
Hb must bind oxygen in lungs and
release it in capillaries
• Adjacent subunits' affinity for oxygen
increases
• This is called positive cooperativity
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
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.
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
Figure 15.34
Oxygen-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.
Fetal hemoglobin has a higher affinity for O2 because
it has a lower affinity for BPG
Sickle cell anemia