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 a Simple Equilibrium Model Explain
Allosteric Kinetics?
4. 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 (enzyme level)
4. Allosteric effectors may be important
5. Enzymes can be modified covalently
6. Zymogens, isozymes and modulator
proteins may play a role
Figure 15.1
Enzymes 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.
Phosphorylation
Adenylylation
ADP-ribosylation
• 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 (enzyme level)
4. Allosteric effectors may be important
5. Enzymes can be modified covalently
6. Zymogens, isozymes and modulator
proteins may play a role
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
Rich in negative
charge
formation of a blood clot.
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.
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 won’t be wasted.
• Modulator proteins are another way that
cells mediate metabolic activity
– cAMP-dependent protein kinase
– Phosphoprotein phosphatase inhibitor-I
Figure 15.6
Cyclic 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.
Figure 15.1
Enzymes 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.
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
• E, 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 Michaelis-Menten
equation
– Their v versus [S] plots yield sigmoid- or Sshaped curve
– A second-order (or higher) relationship between v
and [S]
– Substrate binding is cooperative
Figure 15.7 Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot
characteristic of normal Michaelis - Menten-type enzyme kinetics.
• 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
5. The regulatory effects exerted on the enzyme’s
activity are achieved by comformational changes
occurring in the proetin when effector metabolites
bind
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
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 Rconformers in the total population (that is,
more protein molecules in the R
conformational state).
• 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
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 12:92.)
• 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
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.
•
K system and V system are two different
forms of the MWC model
1. In K system:
– The concentration of S giving half-maximal
velocity, defined as K0.5, changes in response
to effectors
– Vmax is constant
2. In V system
– K0.5 is constant
– Vmax change
– V versus [S] plots are hyperbolic rather than
S-shaped
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
Figure 15.12 The glycogen phosphorylase reaction.
Figure 15.13
The phosphoglucomutase
reaction.
• 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)
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.
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)
Figure 15.15
v 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.
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.
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)
Figure 15.17
In 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 Nterminal 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 22:199-232.)
Figure 15.18
The hormone-activated enzymatic cascade that leads to activation of glycogen
phosphorylase.
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
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.
Figure 15.20
Hormone (H) binding to its receptor (R) creates a
hormone;receptor complex (H:R) that catalyzes
GDP-GTP exchange on the  -subunit of the
heterotrimer G protein (Gbg ), replacing GDP with
GTP. The G -subunit with GTP bound dissociates
from the bg -subunits and binds to adenylyl
cyclase (AC). AC becomes active upon
association with G :GTP and catalyzes the
formation of cAMP from ATP. With time, the
intrinsic GTPase activity of the G -subunit
hydrolyzes the bound GTP, forming GDP; this
leads to dissociation of G :GDP from AC,
reassociation of G with the bg subunits, and
cessation of AC activity. AC and the hormone
receptor H are integral plasma membrane
proteins; G and Gbg are membrane-anchored
proteins.
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 s of 141 residues, 2 bs of 146
Figure 15.21
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.