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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?
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
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 15.4
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
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 wont be wasted.
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.
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
Figure 15.7 Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot
characteristic of normal Michaelis - Menten-type enzyme kinetics.
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).
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.)
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.
Figure 15.13
The phosphoglucomutase
reaction.
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
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.
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 (Gbg ), 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
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
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.