Chapter 5 Amino Acids, Peptides, and Proteins
`
`157
`
`15. Sequence Determination of the Brain Pep-
`tide Leucine Enkephalin A group of peptides that
`influence nerve transmission in certain parts of the
`brain has been isolated from normal brain tissue.
`
`These peptides are knownas opioids, because they
`bind to specific receptors that also bind opiate drugs,
`such as morphine and naloxone. Opioids thus mimic
`some of the properties of opiates. Some researchers
`consider these peptides to be the brain’s own pain
`killers. Using the information below, determine the
`amino acid sequence ofthe opioid leucine enkephalin.
`Explain how your structure is consistent with each
`piece of information.
`
`(a) Complete hydrolysis by 6 M HCl at 110 °C fol-
`lowed by amino acid analysis indicated the presence of
`Gly, Leu, Phe, and Tyr, in a 2:1:1:1 molarratio.
`
`(b) Treatment of the peptide with 1-fluoro-2,4-
`dinitrobenzene followed by complete hydrolysis and
`chromatography indicated the presence of the 2,4-
`dinitrophenyl derivative of tyrosine. No free tyrosine
`could be found.
`
`(c) Complete digestion of the peptide with pepsin
`followed by chromatography yielded a dipeptide con-
`taining Phe and Leu, plus a tripeptide containing Tyr
`and Gly in a 1:2 ratio.
`
`16. Structure of a Peptide Antibiotic from Bacil-
`lus brevis Extracts from the bacterium Bacillus
`
`brevis contain a peptide with antibiotic properties.
`This peptide forms complexes with metal ions and
`apparently disrupts ion transport across the cell
`membranes of other bacterial species, killing them. The
`structure of the peptide has been determined from the
`following observations.
`
`(a) Complete acid hydrolysis of the peptide fol-
`lowed by amino acid analysis yielded equimolar
`amounts of Leu, Orn, Phe, Pro, and Val. Ornis or-
`nithine, an amino acid not present in proteins but pre-
`sent in some peptides. It has the structure
`
`H
`*
`|
`H,N—CH,—CH,—CH,—C—COO-
`*NH,
`
`(b) The molecular weightof the peptide was esti-
`mated as about 1,200.
`
`(c) The peptide failed to undergo hydrolysis when
`treated with the enzyme carboxypeptidase. This en-
`zyme catalyzes the hydrolysis of the carboxyl-terminal
`residue of a polypeptide unless the residue is Pro or
`does not contain a free carboxyl group for some rea-
`son.
`
`(d) Treatment of the intact peptide with 1-fluoro-
`2,4-dinitrobenzene, followed by complete hydrolysis
`and chromatography, yielded only free amino acids
`and the following derivative:
`
`O,N
`
`i
`NO,
`NH—CH,—CH,—CH,—C—COO-
`*NH,
`
`(Hint: Note that the 2,4-dinitrophenyl derivative in-
`volves the amino groupof a side chain rather than the
`qa-amino group.)
`
`(e) Partial hydrolysis of the peptide followed by
`chromatographic separation and sequence analysis
`yielded the following di- and tripeptides (the amino-
`terminal aminoacid is always at theleft):
`
`Leu—Phe Phe-Pro Orn-Leu Val—Orn
`Val—Orn—Leu Phe—Pro-Val
`Pro—Val—Orn
`
`Given the above information, deduce the amino acid
`sequence of the peptide antibiotic. Show your reason-
`ing. When you have arrived at a structure, demon-
`strate that it is consistent with each experimental ob-
`servation.
`
`17. Efficiency in Peptide Sequencing A peptide
`with the primary structure Lys—Arg—Pro—Leu—Ile—
`Asp—Gly—Ala is sequenced by the Edman procedure.
`If each Edman cycle were 96% efficient, what per-
`centage of the amino acids liberated in the fourth cy-
`cle would be leucine? Do the calculation a second
`time, but assume a 99% efficiency for each cycle.
`
`18. Biochemistry Protocols: Your First Protein
`Purification As the newest and least experienced
`student in a biochemistry research lab, your first few
`weeks are spent washing glassware and labeling test
`tubes. You then graduate to making buffers and stock
`solutions for use in various laboratory procedures. Fi-
`nally, you are given responsibility for purifying a pro-
`tein. It is a citric acid cycle enzyme, citrate synthase,
`located in the mitochondrial matrix. Following a pro-
`tocol for the purification, you proceed through the
`steps below. As you work, a more experienced student
`questions you about the rationale for each procedure.
`Supply the answers. (Hint: See Chapter 2 for informa-
`tion on separation of organelles from cells, and Chap-
`ter 4 for information about osmolarity).
`
`(a) You pick up 20 kg of beef hearts from a nearby
`slaughterhouse. You transport the hearts on ice, and
`perform each step of the purification in a walk-in cold
`roomor on ice. You homogenize the beef hearttissue
`ina high-speed blender in a medium containing ~0.2 M
`sucrose, buffered to a pH of 7,2. Why do you use beef
`heart tissue, and in such large quantity? What is
`the purpose of keeping the tissue cold and sus-
`pending it in 0.2 mM sucrose, at pH 7.2? What hap-
`pens to the tissue when it is homogenized?
`
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`

`
`
`Protein Function
`
`Knowing the three-dimensional structure of a protein is an important part
`of understanding how the protein functions. However, the structure shown
`in two dimensions on a pageis deceptively static. Proteins are dynamic mol-
`ecules whose functions almost invariably depend on interactions with other
`molecules, and these interactions are affected in physiologically important
`ways by sometimes subtle, sometimes striking changes in protein confor-
`mation.
`In this chapter, we explore how proteins interact with other molecules
`and how their interactions are related to dynamic protein structure. The im-
`portance of molecular interactions to a protein’s function can hardly be
`overemphasized. In Chapter 6, we saw that the function of fibrous proteins
`as structural elements of cells and tissues depends on stable, long-term
`quaternary interactions between identical polypeptide chains. As we will
`see in this chapter, the functions of many other proteins involve interac-
`tions with a variety of different molecules. Most of these interactions are
`fleeting, though they may be the basis of complex physiological processes
`such as oxygen transport, immune function, and muscle contraction, the
`topics we examine in detail in this chapter. The proteins that carry out
`these processesillustrate the following key principles of protein function,
`some of which will be familiar from the previous chapter:
`
`The functions of many proteins involve the reversible binding of other
`molecules. A molecule bound reversibly by a protein is called a ligand.
`A ligand may be any kind of molecule, including another protein. The
`transient nature of protein-ligand interactionsis critical to life, allowing
`an organism to respond rapidly and reversibly to changing environ-
`mental and metabolic circumstances.
`
`A ligand bindsat a site on the protein called the binding site, which
`is complementary to the ligand in size, shape, charge, and hydropho-
`bic or hydrophilic character. Furthermore, the interaction is specific:
`the protein can discriminate among the thousandsofdifferent mole-
`cules in its environment and selectively bind only one or a few. A
`given protein may have separate binding sites for several different lig-
`ands. These specific molecular interactions are crucial in maintaining
`the high degree oforderin a living system. (This discussion excludes
`the binding of water, which may interact weakly and nonspecifically
`with many parts of a protein. In Chapter 8, we consider water as a
`specific ligand for many enzymes.)
`
`Proteins are flexible. Changes in conformation may be subtle, reflect-
`ing molecular vibrations and small movements of amino acid residues
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`

`204
`
`Part Il Structure and Catalysis
`
`throughout the protein. A protein flexing in this way is sometimes said
`to “breathe.” Changes in conformation may also be quite dramatic,
`with major segments of the protein structure moving as much as sev-
`eral nanometers. Specific conformational changes are frequently es-
`sential to a protein’s function.
`
`The binding of a protein and ligandis often coupled to a conforma-
`tional changein the protein that makes the binding site more comple-
`mentary to the ligand, permitting tighter binding. The structural adap-
`tation that occurs between protein andligandis called inducedfit.
`
`In a multisubunit protein, a conformational change in one subunit
`often affects the conformation of other subunits.
`
`Interactions between ligands and proteins may be regulated, usually
`through specific interactions with one or more additionalligands.
`These other ligands may cause conformational changesin the protein
`that affect the binding of the first ligand.
`
`Enzymes represent a special case of protein function. Enzymes bind
`and chemically transform other molecules—theycatalyze reactions. The
`molecules acted upon by enzymes are called reaction substrates rather
`than ligands, and the ligand-binding site is called the catalytic site or
`active site. In this chapter we emphasize the noncatalytic functions of pro-
`teins. In Chapter 8 we consider catalysis by enzymes, a central topic in bio-
`chemistry. You will see that the themesof this chapter—binding,specificity,
`and conformational change—are continued in the next chapter, with the
`added elementof proteins acting as reactants in chemical transformations.
`
`Reversible Binding of a Protein to a Ligand:
`Oxygen-Binding Proteins
`Myoglobin and hemoglobin may be the most-studied and best-understood
`proteins. They were the first proteins for which three-dimensional struc-
`tures were determined, and our current understanding of myoglobin and
`hemoglobin is garnered from the work of thousands of biochemists over
`several decades. Most important, theyillustrate almost every aspectof that
`most central of biochemical processes: the reversible binding ofa ligand to
`a protein. This classic model of protein function will tell us a great. deal
`about howproteins work.
`
`Oxygen Can Be Bound to a HemeProsthetic Group
`Oxygenis poorly soluble in aqueoussolutions (see Table 4-3) and cannot be
`carried to tissues in sufficient quantity if it is simply dissolved in blood serum.
`Diffusion of oxygen through tissuesis also ineffective over distances greater
`than a few millimeters. The evolutionof larger, multicellular animals depended
`on the evolution of proteins that could transport and store oxygen. How-
`ever, none of the amino acidside chains in proteins is suited for the revers-
`ible binding of oxygen molecules. This role is filed by certain transition met-
`als, among them iron and copper, that have a strong tendency to bind oxygen.
`Multicellular organisms exploit the properties of metals, most commonlyiron,
`for oxygen transport. However, free iron promotes the formation of highly
`reactive oxygen species such as hydroxyl radicals that can damage DNA
`and other macromolecules. Iron used in cells is therefore bound in forms
`that sequester it and/or makeit less reactive. In multicellular organisms—
`especially those in which iron, in its oxygen-carrying capacity, must be
`transported overlarge distances—ironis often incorporated into a protein-
`boundprosthetic group called heme.(A prosthetic group is a compoundper-
`manently associated with a protein that contributes to the protein's function.)
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`Chapter 7 Protein Function
`
`205
`
`figure 7-1
`Heme. The heme group is present in myoglobin, hemo-
`globin, and many other proteins, designated heme pro-
`teins. Hemeconsists of a complex organic ring structure,
`protoporphyrin IX, to which is bound an iron atom in its
`ferrous (Fe**) state. Porphyrins, of which protoporphyrin
`IX is only one example, consist of four pyrrole rings linked
`by methene bridges (a), with substitutions at one or more
`of the positions denoted X. Two representations of heme
`are shownin (b) and (c). The iron atom of heme has six
`coordination bonds: four in the plane of, and bonded
`to, the flat porphyrin ring system, and two perpendicular
`to it (d).
`
`xX
`
`xX
`
`xX
`
`x
`
`x
`
`bi
`
`(a)
`
`x
`
`xX
`
`oO
`
`O-
`
`Heme (or haem) consists of a complex organic ring structure, proto-
`porphyrin, to which is bounda single iron atominits ferrous (Fe’*) state
`(Fig. 7-1). The iron atom has six coordination bonds, four to nitrogen
`atoms that are part of the flat porphyrin ring system and two perpendic-
`ular to the porphyrin. The coordinated nitrogen atoms (which have an
`electron-donating character) help prevent conversion of the heme iron to
`the ferric (Fe®*) state. Iron in the Fe** state binds oxygenreversibly; in the
`Fe?* state it does not bind oxygen. Hemeis found in a numberof oxygen-
`transporting proteins, as well as in some proteins, such as the cytochromes,
`that participate in oxidation-reduction (electron transfer) reactions (Chap-
`ter 19).
`In free heme molecules, reaction of oxygen at one of the two “open” co-
`ordination bonds of iron (perpendicular to the plane of the porphyrin mol-
`ecule, above and below) canresult in irreversible conversion of Fe** to
`Fe**. In heme-containing proteins, this reaction is prevented by sequester-
`ing the heme deepwithin a protein structure where access to the two open
`coordination bondsis restricted. One of these two coordination bondsis oc-
`cupied by a side-chain nitrogen of a His residue. The other is the binding
`site for molecular oxygen (O.) (Fig. 7-2). When oxygen binds, the elec-
`tronic properties of hemeiron change; this accounts for the changein color
`from the dark purple of oxygen-depleted venous blood to the bright red of
`oxygen-rich arterial blood. Some small molecules, such as carbon monoxide
`(CO) and nitric oxide (NO), coordinate to heme iron with greater affinity
`than does O.. When a molecule of CO is bound to heme, Og is excluded,
`which is why COis highly toxic to aerobic organisms. By surrounding and
`sequestering heme, oxygen-binding proteins regulate the access of CO and
`other small molecules to hemeiron.
`
`
`
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`Histidine
`residue
`
`Plane of
`porphyrin
`ring system
`
`N
`fi
`Pie
`CH
`ie
`eet
`C
`C
`PEO Fo Bee OS
`cH,-c
`~o
`‘c—cH,
`‘Cc
`K
`Il.
`¥ df
`gee, oe
`CH
`Feo
`oH
`C—N
`*N=C
`cH |
`\o_cH
`|
`CH,
`¢
`CH,
`
`CH
`
`¢
`CHS
`CH,
`
`(b)
`
`(c)
`
`figure 7-2
`The heme group viewed from the side. This view shows
`the two coordination bonds to Fe** perpendicular to the
`porphyrin ring system. Oneof these two bondsis occu-
`pied by a His residue, sometimes called the proximal His.
`The other is the bindingsite for oxygen. The remaining
`four coordination bonds are in the plane of, and bonded
`to, the flat porphyrin ring system.
`
`
`
`

`

`206
`
`Part ||
`
`Structure and Catalysis
`
`Myoglobin Has a Single Binding Site for Oxygen
`Myoglobin (M/, 16,700; abbreviated Mb)
`is a relatively simple oxygen-
`binding protein found in almost all mammals, primarily in muscle tissue. It
`is particularly abundantin the muscles of diving mammals such as seals and
`whales that must store enough oxygen for prolonged excursions undersea.
`Proteins very similar to myoglobin are widelydistributed, occurring evenin
`some single-celled organisms. Myoglobin stores oxygen for periods when
`energy demandsare high and facilitates its distribution to oxygen-starved
`tissues.
`
`Myoglobin is a single polypeptide of 153 aminoacid residues with one
`molecule of heme. It is typical of the family of proteins called globins,
`which havesimilar primary andtertiary structures. The polypeptide is made
`up of eight a-helical segments connected by bends (Fig. 7-3). About 78%
`of the amino acid residues in the protein are foundin these o helices.
`Any detailed discussion of protein function inevitably involves protein
`structure. Our treatment of myoglobin will be facilitated by introducing
`some structural conventions peculiar to globins. As seen in Figure 7-3, the
`helical segments are labeled A through H. An individual amino acid residue
`may be designated either by its position in the amino acid sequence or by
`its location within the sequence of a particular a-helical segment. For ex-
`ample, the His residue coordinated to the heme in myoglobin, His” (the
`93rd amino acid residue from the amino-terminal end of the myoglobin
`polypeptide sequence), is also called His F8 (the 8th residue in @ helix F).
`The bendsin the structure are labeled AB, CD, EF, and so forth, reflecting
`the a-helical segments they connect.
`
`Protein-Ligand Interactions Can Be Described Quantitatively
`The function of myoglobin depends on the protein’s ability not only to bind
`oxygen, but also to release it when and whereit is needed. Function in bio-
`chemistry often revolves around a reversible protein-ligand interaction of
`this type. A quantitative description of this interaction is therefore a central
`part of many biochemical investigations.
`
`
`
`figure 7-3
`The structure of myoglobin. The eight a-helical seg-
`ments (shown here as cylinders) are labeled A through H.
`Nonhelical residues in the bends that connect them are
`labeled AB, CD, EF, and so forth, indicating the segments
`they interconnect. A few bends, including BC and DE,are
`abrupt and do not contain any residues; these are not
`normally labeled. (The short segmentvisible between D
`andEis an artifact of the computer representation.) The
`hemeis bound in a pocket made up largely of the E and
`F helices, although amino acid residues from other seg-
`ments of the protein also participate.
`
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`Chapter 7 Protein Function
`
`207
`
`In general, the reversible binding of a protein (P) to a ligand (L) can be
`described by a simple equilibrium expression:
`
`P+L = PL
`
`(7-1)
`
`The reaction is characterized by an equilibrium constant, K,, such that
`
`Lee«> TPL]
`(7-2)
`The term K,is an association constant (not to be confused with the K,
`that denotes an acid dissociation constant; see p. 98). The association con-
`stant provides a measureof the affinity of the ligand L for the protein. K,
`has units of M~!; a higher value of K, correspondsto a higheraffinity of the
`ligand for the protein. A rearrangement of Equation 7-2 showsthat the ra-
`tio of bound to free protein is directly proportional to the concentration of
`free ligand:
`
`(7-3)
`
`[PL]
`(P|
`K,[L]
`Whenthe concentration ofthe ligand is much greater than the concentration
`of ligand-bindingsites, the binding of theligand by the protein does not ap-
`preciably change the concentration of free (unbound) ligand—thatis, [L] re-
`mains constant. This condition is broadly applicable to mostligands that bind
`to proteins in cells and simplifies our description of the binding equilibrium.
`Thus we can consider the binding equilibrium from the standpoint of
`the fraction, @ (theta), of ligand-binding sites on the protein that are occu-
`pied by ligand:
`
`a binding sites occupied ——_[PL]
`total binding sites
`[PL] + [P]
`ie
`Substituting K,[L][P] for [PL] (see Eqn 7-3) and rearranging terms gives
`
`6
`
`K,{LI[P]
`
`KL]
`
`__
`
`[4
`
`figure 7-4
`Graphical representations of ligand binding. The frac-
`| ALP) + OP) KIL) +1 ey Aa
`tion of ligand-binding sites occupied, 6, is plotted against
`the concentration of free ligand. Both curves are rectan-
`gular hyperbolas. (a) A hypothetical binding curvefor a
`The term K, can be determined fromaplot of @ versus the concentration of
`ligand L. The LL] at which half of the available ligand-
`free ligand,
`[L] (Fig. 7-4a). Any equation of the form x = y/(y + 2) de-
`binding sites are occupied is equivalent to 1/K,, or Ky.
`scribes a hyperbola, and 6 is thus found to be a hyperbolic function of [L].
`The curve has a horizontal asymptote at @ = 1 and a ver-
`The fraction of ligand-binding sites occupied approaches saturation asymp-
`tical asymptote (not shown) at [L] = —1/K,. (b) A curve
`describing the binding of oxygen to myoglobin. The partial
`totically as [L] increases. The[L] at which half of the available ligand-bind-
`pressure of O, in the air above the solution is expressed
`ing sites are occupied (at @ = 0.5) correspondsto 1/K,.
`in terms of kilopascals (kPa). Oxygen binds tightly to myo-
`globin with a Pep of only 0.26 kPa.
`
`(7-5)
`
`
`
`Ki
`
`5
`({L] (arbitrary units)
`(a)
`
`10
`
`
`
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`1.0
`
`a
`
`5
`pO» (kPa)
`(b)
`
`10
`
`

`

`208
`
`Part Il Structure and Catalysis
`
`It is sometimes intuitively simpler to consider the dissociation con-
`stant, Ky, which is the reciprocal of K, (Ky = 1/K,) andis given in units of
`molar concentration (m). A, is the equilibrium constant for the release of
`ligand. The relevant expressions change to
`
`,-
`[PL] = Ie
`d
`_ (Ly
`(7-8)
`= (L] + K,
`When[L] is equal to K,, half of the ligand-binding sites are occupied.
`When [L] is lower than Ky,little ligand binds to the protein. In order for 90%
`of the available ligand-binding sites to be occupied, [L] must be nine times
`greater than q. In practice, Ay is used much moreoften than K, to express
`the affinity of a protein for a ligand. Note that a lower value of K, corre-
`spondsto a higheraffinity of ligand for the protein. The mathematics can be
`reduced to simple statements: K, is the molar concentration of ligand at
`which half of the available ligand-binding sites are occupied. At this point,
`the protein is said to have reached half saturation with respect to ligand
`binding. The moretightly a protein binds a ligand, the lower the concentra-
`tion of ligand required for half the binding sites to be occupied, and thus the
`lowerthe value of K,. Some representative dissociation constants are given
`in Table 7-1.
`The binding of oxygen to myoglobin follows the patterns discussed
`above, but because oxygenis a gas, we must make some minor adjustments
`to the equations. We can simply substitute the concentration of dissolved
`oxygen for [L] in Equation 7-8 to give
`niug rele
`[02] + Ky
`Asfor any ligand, K, is equal to the [O,] at which half of the available ligand-
`binding sites are occupied, or [O2],;. Equation 7-9 becomes
`
`[O.]
`[92] + [Oz]os
`
`0-8
`(7-7)
`
`(7-9)
`
`(7-10)
`
`=
`
`| SomeProtein Dissociation Constants
`
`| Protein
`Ligand
`Kg ()*
`
`
`| Avidin (egg white)’
`|
`Insulin receptor (human)
`| Anti-HIV immunoglobulin
`|
`(human)?
`| Nickel-binding protein (E. coli)
`1x 10°’
`Calmodulin (rat)$
`3 x 1076
`'
`2x 105
`
`
`
`Biotin
`Insulin
`gp41 (HIV-1 surface
`protein)
`Ni?*
`Gat
`
`1x 10°15
`1.x 107°
`4x 10719
`
`
`
` ARIPOSOLEILEEE RSE SAAR DARRELLRADEEASR ey {DOERR EE CEECERES PEO
`
`“A reported dissociation constantis valid only for the particular solution conditions under which
`it was measured. Kj values for a protein-ligand interaction can be altered, sometimes by several
`orders of magnitude, by changesin solution salt concentration, pH, or other variables.
`"Interaction of avidin with the enzymatic cofactorbiotin is among the strongest noncovalent
`biochemical interactions known.
`‘This immunoglobulin was isolated as part of an effort to develop a vaccine against HIV.
`Immunoglobulins (described later in the chapter) are highly variable, and the K, reported here
`should not be considered characteristic of all immunoglobulins.
`‘Calmodulin has four binding sites for calcium. The values shown reflect the highest- and
`lowest-affinity binding sites observed in one set of measurements.
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`

`Chapter 7 Protein Function
`
`209
`
`The concentration of a volatile substance in solution, however, is always
`proportional to its partial pressure in the gas phase above the solution. In
`experiments using oxygen as a ligand, it is the partial pressure of oxygen,
`pOs, that is varied becausethis is easier to measure than the concentration
`of dissolved oxygen. If we define the partial pressure of oxygen at [Oy]o, as
`Pz», substitution in Equation 7-10 gives
`
`As pO,
`pO, + Pro
`A binding curve for myoglobin that relates @ to pO, is shown in Figure 7—4b.
`
`(7-11)
`
`figure 7-5
`Steric effects on the binding of ligands to the heme of
`myoglobin.
`(a) Oxygen binds to heme with the O, axis at
`an angle, a binding conformation readily accommodated
`by myoglobin. (b) Carbon monoxide binds to free heme
`with the CO axis perpendicular to the plane of the por-
`phyrin ring. CO binding to the heme in myoglobin is
`forced to adopt a slight angle because the perpendicular
`arrangementis sterically blocked by His E7, the distal
`His. This effect weakens the binding of CO to myoglobin.
`(c) Another view showing the arrangement of key amino
`acid residues around the heme of myoglobin. The bound
`Oz is hydrogen-bondedto the distal His, His E7 (His®),
`furtherfacilitating the binding of Oz.
`
`
`
`Protein Structure Affects How Ligands Bind
`The binding of a ligand to a protein is rarely as simple as the above equa-
`tions would suggest. The interaction is greatly affected by protein structure
`and is often accompanied by conformational changes. For example, the
`specificity with which heme binds its various ligands is altered when the
`hemeis a component of myoglobin. CO binds to free heme molecules over
`20,000 times better than does O, (the Ky or Psq for CO binding is more than
`20,000 times lower than that for O,) but binds only about 200 times better
`whenthe hemeis bound in myoglobin. The differenceis partly explained by
`steric hindrance. When O, binds to free heme,the axis of the oxygen mole-
`cule is positioned at an angle to the Fe—O bond (Fig. 7—-5a). In contrast,
`when CO binds to free heme, the Fe, C, and O atomslie in a straight line
`(Fig. 7-5b). In both cases, the binding reflects the geometry of hybrid or-
`bitals in each ligand. In myoglobin, His** (His E7), on the O,-binding side of
`the heme, is too far away to coordinate with the hemeiron, butit does in-
`teract with a ligand bound to heme. This residue, called the distal His, does
`not affect the binding of O. (Fig. 7—-5c) but may precludethe linear binding
`of CO, providing one explanation for the diminished binding of CO to heme
`in myoglobin (and hemoglobin). This effect on CO binding is physiologically
`important, because CO is a low-level byproduct of cellular metabolism.
`Otherfactors, not yet well-defined, also seem to modulate the interaction of
`heme with CO in these proteins.
`The binding of O, to the heme in myoglobin also
`depends on molecular motions, or “breathing,” in the
`protein structure. The heme molecule is deeply
`buried in the folded polypeptide, with no direct path
`for oxygen to go from the surrounding solution to the
`ligand-binding site. If the protein wererigid, O2 could
`not enter or leave the-heme pocket at a measurable
`rate. However, rapid molecular flexing of the amino
`acid side chains produces transient cavities in the
`protein structure, and O, evidently makes its way in
`and out by moving through these cavities. Computer
`simulations of rapid structural fluctuations in myo-
`globin suggest that there are many such pathways.
`One major route is provided by rotation of the side
`chain of the distal His (His™), which occurs on a
`nanosecond (10~* s) time scale. Even subtle confor-
`mational changes can be critical for protein activity.
`
`oO
`O
`#
`Il
`°
`C
`x
`Set Fe —@s
`
`|X
`
`
`
`(ce)
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`MSN Exhibit 1012 - Part 3 - Page 8 of 24
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`

`

`210
`
`Part Il Structure and Catalysis
`
`Oxygen Is Transported in Blood by Hemoglobin
`Nearly all the oxygen carried by whole blood in animals is bound andtrans-
`ported by hemoglobin in erythrocytes (red blood cells). Normal human
`erythrocytes are small (6 to 9 um in diameter), biconcave disks. They are
`formed from precursor stem cells called hemocytoblasts. In the matura-
`tion process, the stem cell produces daughtercells that form large amounts
`of hemoglobin and then lose their intracellular organelles—nucleus, mito-
`chondria, and endoplasmic reticulum. Erythrocytes are thus incomplete,
`vestigial cells, unable to reproduce and, in humans, destined to survive for
`only about 120 days. Their main function is to carry hemoglobin, whichis
`dissolvedin the cytosol at a very high concentration (~34% by weight).
`In arterial blood passing from the lungs through the heart to the pe-
`ripheral tissues, hemoglobin is about 96% saturated with oxygen.In the ve-
`nous blood returning to the heart, hemoglobin is only about 64% saturated.
`Thus, each 100 mL of blood passing through a tissue releases about one-
`third of the oxygenit carries, or 6.5 mL of O, gas at atmospheric pressure
`and body temperature.
`Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 7—4b),
`is relatively insensitive to small changes in the concentration of dissolved
`oxygen and so functions well as an oxygen-storage protein. Hemoglobin,
`with its multiple subunits and O.-bindingsites, is better suited to oxygen
`transport. As we will see, interactions between the subunits of a multimeric
`protein can permit a highly sensitive response to small changesin ligand
`concentration.
`Interactions among the subunits in hemoglobin cause
`conformational changesthatalter the affinity of the protein for oxygen. The
`modulation of oxygen binding allows the O,-transport protein to respond to
`changes in oxygen demandbytissues.
`
`Hemoglobin Subunits Are Structurally Similar to Myoglobin
`Hemoglobin (M, 64,500; abbreviated Hb) is roughly spherical, with a diam-
`eter of nearly 5.5 nm. It is a tetrameric protein containing four heme pros-
`thetic groups, one associated with each polypeptide chain. Adult hemoglo-
`bin contains two types of globin, two a chains (141 residues each) and two
`@ chains (146 residues each). Although fewer than half of the amino acid
`residues in the polypeptide sequencesof the a and B subunits areidentical,
`the three-dimensional structures of the two types of subunits are very sim-
`ilar. Furthermore, their structures are very similar to that of myoglobin
`(Fig. 7-6), even though the amino acid sequencesof the three polypeptides
`are identical at only 27 positions (Fig. 7-7). All three polypeptides are
`
`
`
`Myoglobin
`
`8 subunit of
`hemoglobin
`MSN Exhibit 1012 - Part 3 - Page 9 of 24
`MSN Exhibit 1012 - Part 3 - Page 9 of 24
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`
`figure 7-6
`A comparisonof the structures of myoglobin and the 8
`subunit of hemoglobin.
`
`

`

`Hba Hbg
`F
`F
`K
`R
`100 L
`L
`L
`L
`5
`G
`H
`N
`c
`Vv
`L
`L
`L
`Vv
`Vv
`Cc
`T
`Vv
`
`Mb
`
`LEFIsEAIIHV
`
`v
`
`A
`A
`H
`L
`P
`A
`
`A
`H
`H---
`F
`G
`120K
`
`
`
`E T
`
`CS
`E
`P
`Vv
`Q
`A
`A
`Y
`
`P
`120A
`Vv
`H
`A
`8
`L
`D
`
`v
`
`Q V
`
`
`
`
`
`Distal His E7
`
`
`
`LGAI
`
`L
`T
`N
`A
`Vv
`A
`H
`Vv
`D
`D
`M
`P
`N
`A
`so L
`s
`A
`
`F
`8
`D
`G
`L---
`A
`H
`L
`D
`s80N
`L
`K
`G
`T
`r=
`A
`T
`
`
`
`Ss
`E
`L
`
`E19---L
`
`KKK
`
`80G
`
`HHEAE
`
`FL ---L
`
`KPf
`
`aDe
`
`woeot
`
`G1--100
`
`Proximal His
`
`8
`D
`L
`He
`
`
`Cres
`A
`D
`H
`aR te
`L
`L
`R
`H
`Vv
`Vv
`D
`aes
`P
`100P
`v
`E
`N
`N
`
`
` IZROP|
`
`
`
`KA A
`
`bs:
`
`=O
`
`irco>pd
` Al6---E
`
`SHO>TeOo
`
`A
`Bl --20D
`Vv
`
`AGH
`
`
`
`figure 7-7
`The amino acid sequences of whale myoglobin and the
`a and 8 chains of human hemoglobin. Dashed lines
`mark helix boundaries. To align the sequencesoptimally,
`short breaks must be incorporated into both Hb
`sequences where a few amino acids are present in the
`other sequences. With the exception of the missing D
`helix in Hbe, this alignment permits the use of the helix
`lettering convention that emphasizes the commonposi-
`tioning of amino acid residues that are identicalin all
`three structures (shaded). Residues shaded in red are
`conserved in all known globins. Note that a common
`
`letter-and-number designation for amino acids in two or
`three different structures does not necessarily correspond
`to acommonposition in the linear sequence of amino
`acids in the polypeptides. For example, the distal His
`residue is His E7 in all three structures, but corresponds
`to His, His®®, and His®in the linear sequences of Mb,
`Hba, and Hb§, respectively. Nonhelical residues at the
`amino and carboxyl termini, beyond thefirst (A) and
`last (H) a-helical segments, are labeled NA and HC,
`respectively.
`
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`MSN Exhibit 1012 - Part 3 - Page 10 of 24
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`
`
`
`

`

`212
`
`Part Il
`
`Structure and Catalysis
`
` By
`
`ay
`
`figure 7-8
`Dominantinteractions between hemoglobin subunits.
`In this representation, a subunits are light and 8 subunits
`are dark. The strongest subunit interactions, highlighted,
`occur between unlike subunits. When oxygen binds, the
`a8; contact changeslittle, but there is a large change at
`the a;8> contact, with several ion pairs broken.
`
`members of the globin family of proteins. The helix-naming convention de-
`scribed for myoglobin is also appl