The Allosteric Behavior of Hemoglobin Has Both Symmetry MWC Model and Sequential KNF Model Components Oxygen is accessible only to the heme groups of the -chains when hemoglobin is in th
Trang 1transmitted to the subunit interfaces, where they trigger conformational
readjust-ments that lead to the rupture of interchain salt links.
The Oxy and Deoxy Forms of Hemoglobin Represent Two Different
Conformational States
Hemoglobin resists oxygenation (see Figure 15.20) because the deoxy form is
sta-bilized by specific hydrogen bonds and salt bridges (ion-pair bonds) (Figure
15.27) All of these interactions are broken in oxyhemoglobin, as the molecule
sta-bilizes into a new conformation The shift in helix F upon oxygenation leads to
rupture of the Tyr 145:Val 98 hydrogen bond In deoxyhemoglobin, with these
interactions intact, the C-termini of the four subunits are restrained, and this
con-formational state is termed T, the tense or taut form In oxyhemoglobin, these
C-termini have almost complete freedom of rotation, and the molecule is now in
its R, or relaxed, form.
The Allosteric Behavior of Hemoglobin Has Both Symmetry (MWC)
Model and Sequential (KNF) Model Components
Oxygen is accessible only to the heme groups of the -chains when hemoglobin is
in the T conformational state Max Perutz has pointed out that the heme
environ-ment of -chains in the T state is virtually inaccessible because of steric hindrance
by amino acid residues in the E helix This hindrance disappears when the
hemo-globin molecule undergoes transition to the R conformational state Binding of O2
to the -chains is thus dependent on a T-to-R conformational shift, and this shift is
triggered by the subtle changes that occur when O2 binds to the -chain heme
groups Together these observations lead to a model that is partially MWC and
par-tially KNF: O2 binding to one -subunit and then the other leads to sequential
changes in conformation, followed by a switch in quaternary structure at the
Hb⬊2O2state from T to R Thus, the real behavior of this protein is an amalgam of
the two prominent theoretical models for allosteric behavior.
H⫹Promotes the Dissociation of Oxygen from Hemoglobin
Protons, carbon dioxide, and chloride ions, as well as the metabolite
2,3-bisphosphoglycerate (or BPG), all affect the binding of O2by hemoglobin Their
ef-fects have interesting ramifications, which we shall see as we discuss them in turn
De-oxyhemoglobin has a higher affinity for protons than De-oxyhemoglobin Thus, as the
pH decreases, dissociation of O2from hemoglobin is enhanced In simple symbolism,
ignoring the stoichiometry of O2or Hinvolved:
HbO2 H34HbH O2
(a)
(b)
FIGURE 15.27 Salt bridges between different subunits
in human deoxyhemoglobin These noncovalent, electro-static interactions are disrupted upon oxygenation
(a) A focus on those salt bridges and hydrogen bonds
involving interactions between N-terminal and C-terminal residues in the -chains Residues in the
lower center are Arg 1141 (green) with Val 11 (purple), Asp 2126 (orange), Lys 2127 (yellow), and Val 234 (olive); residues at top are Val 193 (yellow) with Tyr 1140 (purple) (b) A focus on those salt bridges
and hydrogen bonds involving C-terminal residues of
-chains:Val 278 (olive) with Tyr 2145 (purple); His 2
146 (light blue) with Asp 294 (orange) and Lys 140 (yellow) (pdb id 2HHB)
A DEEPER LOOK
In deoxyhemoglobin, the six d electrons of the heme Fe2exist as
four unpaired electrons and one electron pair, and five ligands
can be accommodated: the four N-atoms of the porphyrin ring
sys-tem and histidine F8 In this electronic configuration, the iron
atom is paramagnetic and in the high-spin state When the heme
binds O2as a sixth ligand, these electrons are rearranged into
three epairs and the iron changes to the low-spin state and is
diamagnetic This change in spin state allows the bond between
the Fe2 ion and histidine F8 to become perpendicular to the heme plane and to shorten In addition, interactions between the porphyrin N atoms and the iron strengthen Also, high-spin Fe2 has a greater atomic volume than low-spin Fe2because its four
unpaired eoccupy four orbitals rather than two when the elec-trons are paired in low-spin Fe2 So, low-spin iron is less sterically hindered and able to move nearer to the porphyrin plane
Trang 2Expressed another way, His an antagonist of oxygen binding by Hb, and the satu-ration curve of Hb for O2is displaced to the right as acidity increases (Figure 15.28).
This phenomenon is called the Bohr effect, after its discoverer, the Danish
physiol-ogist Christian Bohr (the father of Niels Bohr, the atomic physicist) The effect has important physiological significance because actively metabolizing tissues produce acid, promoting O2release where it is most needed About two protons are taken
up by deoxyhemoglobin The N-termini of the two -chains and the His 146 residues have been implicated as the major players in the Bohr effect (The pKaof
a free amino terminus in a protein is about 8.0, but the pKaof a protein histidine imidazole is around 6.5.) Neighboring carboxylate groups of Asp 94 residues help
stabilize the protonated state of the His 146 imidazoles that occur in
deoxyhemo-globin However, when Hb binds O2, changes in the conformation of -chains upon
Hb oxygenation move the negative Asp function away, and dissociation of the imidazole protons is favored.
CO2Also Promotes the Dissociation of O2from Hemoglobin
Carbon dioxide has an effect on O2 binding by Hb that is similar to that of H, partly because it produces Hwhen it dissolves in the blood:
The enzyme carbonic anhydrase promotes the hydration of CO2 Many of the protons formed upon ionization of carbonic acid are picked up by Hb as O2dissociates The bicarbonate ions are transported with the blood back to the lungs When Hb be-comes oxygenated again in the lungs, His released and reacts with HCO3 to re-form H2CO3, from which CO2is liberated The CO2is then exhaled as a gas.
In addition, some CO2is directly transported by hemoglobin in the form of car-bamate ( ONHCOO) Free -amino groups of Hb react with CO2reversibly:
This reaction is driven to the right in tissues by the high CO2 concentration; the equilibrium shifts the other way in the lungs where [CO2] is low Thus, carbamyla-tion of the N-termini converts them to anionic funccarbamyla-tions, which then form salt links with the cationic side chains of Arg 141 that stabilize the deoxy or T state of
hemoglobin.
R O NH2 CO2 R O NH O COO H
100
80
60
40
20
0
p O2, torr
Myoglobin
Arterial
p O2
Venous
p O2
pH 7.6
pH 7.4
pH 7.2
pH 7.0
pH 6.8
FIGURE 15.28 The oxygen saturation curves for
myoglo-bin and for hemoglomyoglo-bin at five different pH values: 7.6,
7.4, 7.2, 7.0, and 6.8
Trang 3In addition to CO2, Cland BPG also bind better to deoxyhemoglobin than to
oxyhemoglobin, causing a shift in equilibrium in favor of O2release These various
effects are demonstrated by the shift in the oxygen saturation curves for Hb in the
presence of one or more of these substances (Figure 15.29) Note that the O2
-binding curve for Hb BPG CO2fits that of whole blood very well.
2,3-Bisphosphoglycerate Is an Important Allosteric Effector
for Hemoglobin
The binding of 2,3-bisphosphoglycerate (BPG) to Hb promotes the release
of O2(Figure 15.29) Erythrocytes (red blood cells) normally contain about 4.5
mM BPG, a concentration equivalent to that of tetrameric hemoglobin molecules.
Interestingly, this equivalence is maintained in the Hb⬊BPG binding
stoichiome-try because the tetrameric Hb molecule has but one binding site for BPG This
site is situated within the central cavity formed by the association of the four
sub-units The strongly negative BPG molecule (Figure 15.30) is electrostatically bound
via interactions with the positively charged functional groups of each Lys 82, His
2, His 143, and the NH3 -terminal group of each -chain These positively
charged residues are arranged to form an electrostatic pocket complementary to
the conformation and charge distribution of BPG (Figure 15.31) In effect, BPG
crosslinks the two -subunits The ionic bonds between BPG and the two -chains
aid in stabilizing the conformation of Hb in its deoxy form, thereby favoring the
dissociation of oxygen In oxyhemoglobin, this central cavity is too small for BPG
to fit Or, to put it another way, the conformational changes in the Hb molecule
that accompany O2 binding perturb the BPG-binding site so that BPG can no
longer be accommodated Thus, BPG and O2are mutually exclusive allosteric
ef-fectors for Hb, even though their binding sites are physically distinct.
BPG Binding to Hb Has Important Physiological Significance
The importance of the BPG effect is evident in Figure 15.29 Hemoglobin stripped
of BPG is virtually saturated with O2at a pO2of only 20 torr, and it cannot release its
oxygen within tissues, where the pO2is typically 40 torr BPG shifts the oxygen
satu-ration curve of Hb to the right, making the Hb an O2delivery system eminently
suited to the needs of the organism BPG serves this vital function in humans, most
primates, and a number of other mammals However, the hemoglobins of cattle,
sheep, goats, deer, and other animals have an intrinsically lower affinity for O2, and
these Hbs are relatively unaffected by BPG
Fetal Hemoglobin Has a Higher Affinity for O2Because
It Has a Lower Affinity for BPG
The fetus depends on its mother for an adequate supply of oxygen, but its circulatory
system is entirely independent Gas exchange takes place across the placenta Ideally
20
0
p O2, torr
100
80
60
40
Stripped Hb
Hb+ CO2
Hb+ BPG
Hb+ BPG + CO2 Whole blood
FIGURE 15.29 Oxygen-binding curves of blood and of hemoglobin in the absence and presence of CO2and BPG From left to right: stripped Hb, Hb CO2, Hb BPG, Hb BPG CO2, and whole blood
C
P O
H2C OPO3–
O–
O–
O C H H C H C O
O O
–O –O
–
FIGURE 15.30 The structure, in ionic form, of BPG or 2,3-bisphosphoglycerate, an important allosteric effector
for hemoglobin
Trang 4then, fetal Hb should be able to absorb O2better than maternal Hb so that an effective transfer of oxygen can occur Fetal Hb differs from adult Hb in that the -chains are
replaced by very similar, but not identical, 146-residue subunits called -chains (gamma
chains) Fetal Hb is thus 22 Recall that BPG functions through its interaction with the -chains BPG binds less effectively with the -chains of fetal Hb (also called Hb F).
(Fetal -chains have Ser instead of His at position 143 and thus lack two of the positive
charges in the central BPG-binding cavity.) Figure 15.32 compares the relative affinities
of adult Hb (also known as Hb A) and Hb F for O2under similar conditions of pH and [BPG] Note that Hb F binds O2at pO2values where most of the oxygen has dissoci-ated from Hb A Much of the difference can be attributed to the diminished capacity
of Hb F to bind BPG (compare Figures 15.29 and 15.32); Hb F thus has an intrinsically greater affinity for O2, and oxygen transfer from mother to fetus is ensured.
Sickle-Cell Anemia Is Characterized by Abnormal Red Blood Cells
In 1904, a Chicago physician treated a 20-year-old black college student complaining
of headache, weakness, and dizziness The blood of this patient revealed serious anemia—only half the normal number of red cells were present Many of these cells
100
80
60
40
20
pO2, torr
Hb F
Hb A
FIGURE 15.32 Comparison of the oxygen saturation
curves of Hb A and Hb F under similar conditions of pH
and [BPG]
FIGURE 15.31 The ionic binding of BPG to the two
-subunits of Hb BPG lies at center of the cavity
be-tween the two -subunits.The highlighted residues are
N-terminal Val 1and Val 2(yellow), His 12, His 22,
His1143, and His 2143 (purple), Lys 182 and
Lys 282 (green) (pdb id 1B86)
Trang 5were abnormally shaped; in fact, instead of the characteristic disc shape, these
ery-throcytes were elongated and crescentlike in form, a feature that eventually gave
name to the disease sickle-cell anemia These sickle cells pass less freely through the
capillaries, impairing circulation and causing tissue damage Furthermore, these cells
are more fragile and rupture more easily than normal red cells, leading to anemia.
Sickle-Cell Anemia Is a Molecular Disease
A single amino acid substitution in the -chains of Hb causes sickle-cell anemia
Re-placement of the glutamate residue at position 6 in the -chain by a valine residue
marks the only chemical difference between Hb A and sickle-cell hemoglobin, Hb S.
The amino acid residues at position 6 lie at the surface of the hemoglobin molecule.
In Hb A, the ionic R groups of the Glu residues fit this environment In contrast, the
aliphatic side chains of the Val residues in Hb S create hydrophobic protrusions
where none existed before To the detriment of individuals who carry this trait, a
hy-drophobic pocket forms in the EF corner of each -chain of Hb when it is in the
de-oxy state, and this pocket nicely accommodates the Val side chain of a neighboring
Hb S molecule (Figure 15.33) This interaction leads to the aggregation of Hb S
mol-ecules into long, chainlike polymeric structures The obvious consequence is that
de-oxyHb S is less soluble than dede-oxyHb A The concentration of hemoglobin in red
blood cells is high (about 150 mg/mL), so even in normal circumstances it is on the
HUMAN BIOCHEMISTRY
Hemoglobin and Nitric Oxide
Nitric oxide (NO) is a simple gaseous molecule whose many
re-markable physiological functions are still being discovered For
ex-ample, NO is known to act as a neurotransmitter and as a second
messenger in signal transduction (see Chapter 32) Furthermore,
endothelial relaxing factor (ERF, also known as
endothelium-derived relaxing factor, or EDRF), an elusive hormonelike agent
that acts to relax the musculature of the walls (endothelium) of
blood vessels and lower blood pressure, has been identified as
NO It has long been known that NO is a high-affinity ligand for
Hb, binding to its heme-Fe2 atom with an affinity 10,000 times
greater than that of O2 An enigma thus arises: Why isn’t NO
in-stantaneously bound by Hb within human erythrocytes and
pre-vented from exerting its vasodilation properties?
The reason that Hb doesn’t block the action of NO is due to
a unique interaction between Cys 93 of Hb and NO discovered
by Li Jia, Celia and Joseph Bonaventura, and Johnathan Stamler at
Duke University Nitric oxide reacts with the sulfhydryl group of
Cys 93, forming an S-nitroso derivative:
This S-nitroso group is in equilibrium with other S-nitroso
com-pounds formed by reaction of NO with small-molecule thiols
such as free cysteine or glutathione (an
isoglutamylcysteinylgly-cine tripeptide):
O CH2O S O N O
These small-molecule thiols serve to transfer NO from erythro-cytes to endothelial receptors, where it acts to relax vascular ten-sion NO itself is a reactive free-radical compound whose biolog-ical half-life is very short (1–5 sec) S-nitrosoglutathione has a half-life of several hours
The reactions between Hb and NO are complex NO forms
a ligand with the heme-Fe2that is quite stable in the absence of
O2 However, in the presence of O2, NO is oxidized to NO3 and the heme-Fe2of Hb is oxidized to Fe3, forming methemoglobin Fortunately, the interaction of Hb with NO is controlled by the allosteric transition between R-state Hb (oxyHb) and T-state Hb (deoxyHb) Cys 93 is more exposed and reactive in R-state Hb than in T-state Hb, and binding of NO to Cys 93 precludes
re-action of NO with heme iron Upon release of O2from Hb in tis-sues, Hb shifts conformation from R state to T state, and binding
of NO at Cys 93 is no longer favored Consequently, NO is
re-leased from Cys 93 and transferred to small-molecule thiols for delivery to endothelial receptors, causing capillary vasodilation This mechanism also explains the puzzling observation that free
Hb produced by recombinant DNA methodology for use as a whole-blood substitute causes a transient rise of 10 to 12 mm Hg
in diastolic blood pressure in experimental clinical trials (Con-ventional whole-blood transfusion has no such effect.) It is now ap-parent that the “synthetic” Hb, which has no bound NO, is bind-ing NO in the blood and preventing its vasoregulatory function
In the course of hemoglobin evolution, the only invariant amino acid residues in globin chains are His F8 (the obligatory heme ligand) and a Phe residue acting to wedge the heme into its pocket However, in mammals and birds, Cys 93 is also invariant,
no doubt due to its vital role in NO delivery
Adapted from Jia, L., et al., 1996 S-Nitrosohaemoglobin: A dynamic
activ-ity of blood involved in vascular control Nature 380:221–226.
O OCH2OCH2OC
OSON O
O
COO
COO
H
N
O
CH2
S-nitrosoglutathione
Trang 6verge of crystallization The formation of insoluble deoxyHb S fibers distorts the red cell into the elongated sickle shape characteristic of the disease.2
2In certain regions of Africa, the sickle-cell trait is found in 20% of the people Why does such a dele-terious heritable condition persist in the population? For reasons as yet unknown, individuals with this trait are less susceptible to the most virulent form of malaria The geographic distribution of malaria and the sickle-cell trait are positively correlated
Oxy-hemoglobin A
β1
β2
α1
α2
β1
β2
α1
α2
β1
β2
α1
α2
β1
β2
α1
α2
Deoxy-hemoglobin A
Oxy-hemoglobin S
Deoxy-hemoglobin S
α1 α2
Deoxyhemoglobin S polymerizes into filaments
α1 α2
α1 α2
β1 β2
α1 α2
β1 β2
α1 α2
β1 β2
α1 α2
ANIMATED FIGURE 15.33 The polymerization of Hb S via the interactions between the hydrophobic Val side chains at position 6 and the hydrophobic pockets in the EF corners of -chains in
neigh-boring Hb molecules (a) The protruding “block” on Oxy S represents the Val hydrophobic protrusion The
com-plementary hydrophobic pocket in the EF corner of deoxy -chains is represented by a square-shaped
indenta-tion (This indentation is probably present in Hb A also.) Only the 2Val protrusions and the 1EF pockets are shown (The 1Val protrusions and the 2EF pockets are not involved, although they are present.) (b) The
polymerization of Hb S via 2Val6 insertions into neighboring 1pockets (c) Molecular graphic of an Hb S
dimer of tetramers.2Val residues are highlighted in blue; heme is shown in red (pdb id 2HBS) (d) Molecular
graphic of the Hb S filament (pdb id 2HBS) See this figure animated at www.cengage.com/login.
SUMMARY
15.1 What Factors Influence Enzymatic Activity? The two prominent
ways to regulate enzyme activity are (1) to increase or decrease the
num-ber of enzyme molecules or (2) to increase or decrease the intrinsic
activity of each enzyme molecule Changes in enzyme amounts are
typ-ically regulated via gene expression and protein degradation Changes
in the intrinsic activity of enzyme molecules are achieved principally by
allosteric regulation or covalent modification
15.2 What Are the General Features of Allosteric Regulation?
Allosteric enzymes show a sigmoid response of velocity, v, to increasing
[S], indicating that binding of S to the enzyme is cooperative Allosteric
enzymes often are susceptible to feedback inhibition Allosteric enzymes
may also respond to allosteric activation Allosteric activators signal a
need for the end product of the pathway in which the allosteric enzyme
functions As a general rule, allosteric enzymes are oligomeric, with each
monomer possessing a substrate-binding site and an allosteric site where
effectors bind Interaction of one subunit of an allosteric enzyme with its
substrate (or its effectors) is communicated to the other subunits of the enzyme through intersubunit interactions These interactions can lead
to conformational transitions that make it easier (or harder) for addi-tional equivalents of ligand (S, A, or I) to bind to the enzyme
15.3 Can Allosteric Regulation Be Explained by Conformational Changes in Proteins? Monod, Wyman, and Changeux postulated that the subunits of allosteric enzymes can exist in two conformational states (R and T), that all subunits in any enzyme molecule are in the
same conformational state (symmetry), that equilibrium strongly favors
the T conformational state, and that S binds preferentially (“only”) to the R state Sigmoid binding curves result, provided that [T0] [R0]
in the absence of S and that S binds “only” to R Positive or negative effectors influence the relative T/R equilibrium by binding preferen-tially to T (negative effectors) or R (positive effectors), and the sub-strate saturation curve is shifted to the right (negative effectors) or left (positive effectors)
Trang 7In an alternative allosteric model suggested by Koshland, Nemethy,
and Filmer (the KNF model), S binding leads to conformational changes
in the enzyme The altered conformation of the enzyme may display
higher affinity for the substrate (positive cooperativity) or lower affinity
for the substrate or other ligand (negative cooperativity) Negative
co-operativity is not possible within the MWC model Reversible changes in
the oligomeric state of a protein can also yield allosteric behavior For
ex-ample, a monomer–oligomer equilibrium for an allosteric protein,
where only the oligomer binds S and [monomer] [oligomer], would
show cooperative substrate binding
15.4 What Kinds of Covalent Modification Regulate the Activity of
Enzymes? Reversible phosphorylation is the most prominent form of
covalent modification in cellular regulation Phosphorylation is
accom-plished by protein kinases; phosphoprotein phosphatases act in the
reverse direction to remove the phosphate group Regulation must be
imposed on these converter enzymes so that their enzyme targets adopt
the metabolically appropriate state (active versus inactive) Thus, these
converter enzymes are themselves the targets of allosteric regulation or
covalent modification Although several hundred chemical
modifica-tions of proteins have been described, only a few are used for reversible
conversion of enzymes between active and inactive forms Besides
phos-phorylation, these regulatory types include adenylylation, uridylylation,
ADP-ribosylation, methylation, and oxidation-reduction of protein
disulfide bonds
15.5 Is the Activity of Some Enzymes Controlled by Both Allosteric
Regulation and Covalent Modification? Some enzymes are subject to
both allosteric regulation and regulation by covalent modification A
prime example is glycogen phosphorylase Glycogen phosphorylase
exists in two forms, a and b, which differ only in whether or not Ser14
-OH is phosphorylated (a) or not (b) Glycogen phosphorylase b shows
positive cooperativity in binding its substrate, phosphate In addition,
glycogen phosphorylase b is allosterically activated by the positive
effec-tor AMP In contrast, ATP and glucose-6-P are negative effeceffec-tors for
glycogen phosphorylase b Covalent modification of glycogen phospho-rylase b by phosphophospho-rylase kinase converts it from a less active, allosteri-cally regulated form to the more active a form that is less responsive to
allosteric regulation Glycogen phosphorylase is both activated and freed from allosteric control by covalent modification
Special Focus: Is There an Example in Nature That Exemplifies the Relationship Between Quaternary Structure and the Emergence of Al-losteric Properties? Hemoglobin and Myoglobin—Paradigms of Pro-tein Structure and Function Myoglobin and hemoglobin have illumi-nated our understanding of protein structure and function Myoglobin
is monomeric, whereas hemoglobin has a quaternary structure Myo-globin functions as an oxygen-storage protein in muscle; Hb is an O2 -transport protein When Mb binds O2, its heme iron atom is drawn within the plane of the heme, slightly shifting the position of the F helix
of the protein Hemoglobin shows cooperative binding of O2and allo-steric regulation by H, CO2, and 2,3-bisphosphoglycerate The allosteric properties of Hb can be traced to the movement of the F helix upon O2
binding to Hb heme groups and the effects of F-helix movement on in-teractions between the protein’s subunits that alter the intrinsic affinity
of the other subunits for O2 The allosteric transitions in Hb partially conform to the MWC model in that a concerted conformational change from a T-state, low-affinity conformation to an R-state, high-affinity form takes place after 2 O2are bound (by the 2 Hb -subunits) However, Hb
also behaves somewhat according to the KNF model of allostery in that oxygen binding leads to sequential changes in the conformation and O2
affinity of hemoglobin subunits Sickle-cell anemia is a molecular disease traceable to a tendency for Hb S to polymerize as a consequence of hav-ing a E6V amino acid substitution that creates a “sticky” hydrophobic
patch on the Hb surface
PROBLEMS
Preparing for an exam? Create your own study path for this
chapter at www.cengage.com/login
1.List six general ways in which enzyme activity is controlled
2.Why do you suppose proteolytic enzymes are often synthesized as
inactive zymogens?
3.(Integrates with Chapter 13.) Draw both Lineweaver–Burk plots
and Hanes–Woolf plots for an MWC allosteric enzyme system,
show-ing separate curves for the kinetic response in (a) the absence of
any effectors, (b) the presence of allosteric activator A, and (c) the
presence of allosteric inhibitor I
4.The KNF model for allosteric transitions includes the possibility of
negative cooperativity Draw Lineweaver–Burk and Hanes–Woolf
plots for the case of negative cooperativity in substrate binding (As
a point of reference, include a line showing the classic Michaelis–
Menten response of v to [S].)
allows the calculation of Y (the
fractional saturation of hemoglobin with O2), given P50and n (see
box on page 472) Let P50 26 torr and n 2.8 Calculate Y in the
lungs, where p O2 100 torr, and Y in the capillaries, where pO2
40 torr What is the efficiency of O2delivery under these
condi-tions (expressed as Ylungs Ycapillaries)? Repeat the calculations, but
for n 1 Compare the values for Ylungs Ycapillaries for n 2.8
versus Ylungs Ycapillariesfor n 1 to determine the effect of
coop-erative O2binding on oxygen delivery by hemoglobin
6.The cAMP formed by adenylyl cyclase (Figure 15.18) does not persist
because 5-phosphodiesterase activity prevalent in cells hydrolyzes
cAMP to give 5-AMP Caffeine inhibits 5-phosphodiesterase activity
Describe the effects on glycogen phosphorylase activity that arise as a
consequence of drinking lots of caffeinated coffee
p O2
P50
Y
(1 Y)
7. If no precautions are taken, blood that has been stored for some time becomes depleted in 2,3-BPG What happens if such blood is used in a transfusion?
8. Enzymes have evolved such that their K m values (or K0.5values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate(s) Assume that glycogen phosphorylase is assayed at [Pi]⬇ K0.5in the absence and presence of AMP or ATP Estimate from Figure 15.14 the relative glycogen phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and
(c) ATP is present (Hint: Use a ruler to get relative values for the velocity v at the appropriate midpoints of the saturation curves.)
9. Cholera toxin is an enzyme that covalently modifies the G-subunit
of G proteins (Cholera toxin catalyzes the transfer of ADP-ribose from NADto an arginine residue in G, an ADP-ribosylation reac-tion.) Covalent modification of G inactivates its GTPase activity Predict the consequences of cholera toxin on cellular cAMP and glycogen levels
10. Allosteric enzymes that sit at branch points leading to several essen-tial products sometimes display negative cooperativity for feedback inhibition (allosteric inhibition) by one of the products What might be the advantage of negative cooperativity instead of positive cooperativity in feedback inhibitor binding by such enzymes?
11. Consult Table 15.2 and
a Suggest a consensus amino acid sequence within phosphorylase kinase that makes it a target of protein kinase A (the cAMP-dependent protein kinase)
b Suggest an effective amino acid sequence for a regulatory domain pseudosubstrate sequence that would exert intrasteric control on phosphorylase kinase by blocking its active site
12. What are the relative advantages (and disadvantages) of allosteric regulation versus covalent modification?
Trang 813. You land a post as scientific investigator with a pharmaceutical
com-pany that would like to develop drugs to treat people with sickle-cell
anemia They want ideas from you! What molecular properties of
Hb S might you suggest as potential targets of drug therapy?
14. Under appropriate conditions, nitric oxide (NO) combines with
Cys 93 in hemoglobin and influences its interaction with O2 Is
this interaction an example of allosteric regulation or covalent
modification?
15. Lactate, a metabolite produced under anaerobic conditions in
mus-cle, lowers the affinity of myoglobin for O2 This effect is beneficial,
because O2dissociation from Mb under anaerobic conditions will
provide the muscle with oxygen Lactate binds to Mb at a site
dis-tinct from the O2-binding site at the heme In light of this
observa-tion, discuss whether myoglobin should be considered an allosteric
protein
16.An allosteric model based on multiple oligomeric states of a
pro-tein has been proposed by E K Jaffe (2005 Morpheeins: A new
structural paradigm for allosteric regulation Trends in Biochemical
Sciences 30:490–497) This model coins the term morpheeins to
de-scribe the different forms of a protein that can assume more than
one conformation, where each distinct conformation assembles
into an oligomeric structure with a fixed number of subunits For
example, conformation A of the protein monomer forms trimers,
whereas conformation B of the monomer forms tetramers If
trimers and tetramers have different kinetic properties (Kmand kcat
values), as in low-activity trimers and high-activity tetramers, then
the morpheein ensemble behaves like an allosterically regulated
enzyme Drawing on the traditional MWC model as an analogy,
di-agram a simple morpheein model in which wedge-shaped protein
monomers assemble into trimers but the alternative conformation
for the monomer (a square shape) forms tetramers Further, the substrate, S, or allosteric regulator, A, binds “only” to the square conformation, and its binding prevents the square from adopting the wedge conformation Describe how your diagram yields allo-steric behavior
17.CTP synthetase catalyzes the synthesis of CTP from UTP:
UTP ATP glutamine st CTP glutamate ADP Pi
The substrates UTP and ATP show positive cooperativity in their binding to the enzyme, which is an 4-type homotetramer However, the other substrate, glutamine, shows negative cooperativity Draw
substrate saturation curves of the form v versus [S]/K0.5for each of these three substrates that illustrate these effects
18.Glyceraldehyde-3-phosphate dehydrogenase catalyzes the synthesis
of 1,3-bisphosphoglycerate:
Glyceraldehyde-3-P Pi NADst 1,3-BPG NADH H
The enzyme is a tetramer NADbinding shows negative coopera-tivity Draw a diagram of possible conformational states for this tetrameric enzyme and its response to NADbinding that illustrates negative cooperativity
Preparing for the MCAT Exam
19.On the basis of the graphs shown in Figures 15.28 and 15.29 and the relationship between blood pH and respiration (Chapter 2), predict the effect of hyperventilation and hypoventilation on Hb:O2affinity
20.Figure 15.17 traces the activation of glycogen phosphorylase from
hormone to phosphorylation of the b form of glycogen phosphory-lase to the a form These effects are reversible when hormone
dis-appears Suggest reactions by which such reversibility is achieved
FURTHER READING
General References
Fersht, A., 1999 Structure and Mechanism in Protein Science: A Guide to
En-zyme Catalysis and Protein Folding New York: W H Freeman.
Protein Kinases
Johnson, L., 2007 Protein kinases and their therapeutic exploitation
Transactions 35:7–11.
Manning, G., et al., 2002 The protein kinase complement of the human
genome Science 298:1912–1934 A catalog of the protein kinase
genes identified within the human genome About 2% of all
eukary-otic genes encode protein kinases
Allosteric Regulation
Changeux, J.-P., and Edelstein, S J., 2005 Allosteric mechanisms of
sig-nal transduction Science 308:1424–1428.
Helmstaedt, K., Krappman, S., and Braus, G H., 2001 Allosteric
regula-tion of catalytic activity Escherichia coli aspartate transcarbamoylase
versus yeast chorismate mutase Microbiology and Molecular Biology
Re-views 65:404–421 The authors present evidence to show that the
MWC two-state model is oversimplified, as Monod, Wyman, and
Changeux themselves originally stipulated
Koshland, D E., Jr., and Hamadani, K., 2002 Proteomics and models for
enzyme cooperativity Journal of Biological Chemistry 277:46841–46844.
An overview of both the MWC and the KNF models for allostery and
a discussion of the relative merits of these models The fact that the
number of allosteric enzymes showing negative cooperativity is about
the same as the number showing positive cooperativity is an
impor-tant focus of this review
Koshland, D E., Jr., Nemethy, G., and Filmer, D., 1966 Comparison of
experimental binding data and theoretical models in proteins
con-taining subunits Biochemistry 5:365–385 The KNF model.
Kuriyan, J., and Eisenberg, D., 2007 The origin of protein interactions
and allostery in colocalization Nature 450:983–990.
Monod, J., Wyman, J., and Changeux, J-P., 1965 On the nature of
allo-steric transitions: A plausible model Journal of Molecular Biology 12:
88–118 The classic paper that provided the first theoretical analysis
of allosteric regulation
Schachman, H K., 1990 Can a simple model account for the allosteric
transition of aspartate transcarbamoylase? Journal of Biological
Chem-istry 263:18583–18586 Tests of the postulates of the allosteric
mod-els through experiments on aspartate transcarbamoylase
Swain, J F., and Gierasch, L M., 2006 The changing landscape of
pro-tein allostery Current Opinion in Structural Biology 16:102–108.
Glycogen Phosphorylase
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.
Johnson, L N., and Barford, D., 1994 Electrostatic effects in the
con-trol of glycogen phosphorylase by phosphorylation Protein Science
3:1726–1730
Lin, K., et al., 1996 Comparison of the activation triggers in yeast and
muscle glycogen phosphorylase Science 273:1539–1541.
Lin, K., et al., 1997 Distinct phosphorylation signals converge at the
cat-alytic center in glycogen phosphorylases Structure 5:1511–1523.
Rath, V L., et al., 1996 The evolution of an allosteric site in
phosphory-lase Structure 4:463–473.
Hemoglobin
Ackers, G K., 1998 Deciphering the molecular code of hemoglobin
al-lostery Advances in Protein Chemistry 51:185–253.
Dickerson, R E., and Geis, I., 1983 Hemoglobin: Structure, Function, Evo-lution and Pathology Menlo Park, CA: Benjamin/Cummings.
Henry, E R., et al., 2002 A tertiary two-state allosteric model for
hemo-globin Biophysical Chemistry 98:149–164.
Weiss, J N., 1997 The Hill equation revisited: Uses and abuses The
FASEB Journal 11:835–841.
Trang 9© Bettmann/CORBIS
16.1 What Is a Molecular Motor?
Motor proteins, also known as molecular motors, use chemical energy (ATP) to
or-chestrate movements, transforming ATP energy into the mechanical energy of
mo-tion In all cases, ATP hydrolysis is presumed to drive and control protein
confor-mational changes that result in sliding or walking movements of one molecule
relative to another To carry out directed movements, molecular motors must be able
to associate and dissociate reversibly with a polymeric protein array, a surface or
sub-structure in the cell ATP hydrolysis drives the process by which the motor protein
ratchets along the protein array or surface As fundamental and straightforward as
all this sounds, elucidation of these basically simple processes has been extremely
challenging for biochemists, involving the application of many sophisticated
chemi-cal and physichemi-cal methods in many different laboratories This chapter describes the
structures and chemical functions of molecular motor proteins and some of the
ex-periments by which we have come to understand them
Molecular motors may be linear or rotating Linear motors crawl or creep along a
polymer lattice, whereas rotating motors consist of a rotating element (the “rotor”)
and a stationary element (the “stator”), in a fashion much like a simple electrical
motor The linear motors we will discuss include kinesins and dyneins (which crawl
along microtubules), myosin (which slides along actin filaments in muscle), and
DNA helicases (which move along a DNA lattice, unwinding duplex DNA to form
single-stranded DNA) Rotating motors include the flagellar motor complex,
de-scribed in this chapter, and the ATP synthase, which will be dede-scribed in Chapter 20.
16.2 What Is the Molecular Mechanism
of Muscle Contraction?
Muscle Contraction Is Triggered by Ca2ⴙRelease
from Intracellular Stores
Muscle contraction is the result of interactions between myosin and actin, the two
predominant muscle proteins Thick filaments of myosin slide along thin
fila-ments of actin to cause contraction.
The cells of skeletal muscle are long and multinucleate and are referred to as
muscle fibers. Skeletal muscles in higher animals consist of
100-some as long as the muscle itself Each of these muscle fibers contains hundreds of
myofibrils (Figure 16.1), each of which spans the length of the fiber and is about
Michelangelo’s David epitomizes the musculature of
the human form
Buying bread from a man in Brussels
He was six foot four and full of muscles
I said “Do you speak-a my language?”
He just smiled and gave me a Vegemite sandwich.
Colin Hay and Ron Strykert,
lyrics from Down Under
KEY QUESTIONS 16.1 What Is a Molecular Motor?
16.2 What Is the Molecular Mechanism of Muscle Contraction?
16.3 What Are the Molecular Motors That Orchestrate the Mechanochemistry
of Microtubules?
16.4 How Do Molecular Motors Unwind DNA?
16.5 How Do Bacterial Flagella Use a Proton Gradient to Drive Rotation?
ESSENTIAL QUESTION
Movement is an intrinsic property associated with all living things Within cells,
mole-cules undergo coordinated and organized movements, and cells themselves may move
across a surface At the tissue level, muscle contraction allows higher organisms to
carry out and control crucial internal functions, such as peristalsis in the gut and the
beating of the heart Muscle contraction also enables the organism to perform
orga-nized and sophisticated movements, such as walking, running, flying, and swimming.
How can biological macromolecules, carrying out conformational changes on
the molecular level, achieve these feats of movement that span the microscopic
and macroscopic worlds?
Create your own study path for this chapter with tutorials, simulations, animations,
and Active Figures at www.cengage.com/login.
Trang 10Sarcolemma Sarcoplasmic reticulum Terminal cisternae Transverse tubule
Nucleus Contractile filaments
Myofibril
Mitochondrion
SR membrane Transverse tubule
FIGURE 16.1 The structure of a skeletal muscle cell,
showing the manner in which transverse tubules
en-able the sarcolemmal membrane to extend into the
in-terior of the fiber T-tubules and sarcoplasmic reticulum
(SR) membranes are juxtaposed at structures termed
triad junctions (inset)
HUMAN BIOCHEMISTRY
Smooth Muscle Effectors Are Useful Drugs
Not all vertebrate muscle is skeletal muscle Vertebrate organisms
employ smooth muscle for long, slow, and involuntary contractions
in various organs, including large blood vessels, intestinal walls, the
gums of the mouth, and in the female, the uterus Smooth muscle
contraction is triggered by Ca2-activated phosphorylation of
myosin by myosin light-chain kinase (MLCK) The action of
epi-nephrine and related agents forms the basis of therapeutic control
of smooth muscle contraction Breathing disorders, including
asthma and various allergies, can result from excessive contraction
of bronchial smooth muscle tissue Treatment with epinephrine,
whether by tablets or aerosol inhalation, inhibits MLCK and relaxes
bronchial muscle tissue More specific bronchodilators, such as
albuterol (see accompanying figure), act more selectively on the lungs and avoid the undesirable side effects of epinephrine on the heart Albuterol is also used to prevent premature labor in pregnant women because of its relaxing effect on uterine smooth muscle
Conversely, oxytocin, known also as Pitocin, stimulates contraction
of uterine smooth muscle This natural secretion of the pituitary gland is often administered to induce labor
CH2OH
H
CH3
CH3
Albuterol
Oxytocin (Pitocin)
䊱 The structure of oxytocin.