Lehninger Principles of Biochemistry

1
Chap. 5A Protein Function

• Topics
• Reversible Binding of a Protein to a Ligand
• Oxygen-binding Proteins

• The heme group
• Myoglobin
• Hemoglobin
• Sickle-cell Anemia

Fig. 5-19. Normal (a) and sickle-cell anemia (b)
erythrocytes.
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Intro. to Ligand-binding Proteins
Ligands are molecules that can be bound
reversibly by a protein. Ligands can be any type
of molecule, including another protein. Proteins
that bind ligands do so at sequences called the
binding site. The binding site is complementary
in shape to the ligand that is bound. The degree
of complementarity determines the binding
specificity and strength. Most proteins undergo
conformational changes on binding to a ligand.
Changes can be small (i.e., breathing), or can
be major. In a multisubunit protein, a
conformational change in one subunit can affect
the conformation of other subunits. Enzymes also
bind to complementary molecules. However, in
addition to binding, enzymes chemically transform
these components. The binding site on an enzyme
is referred to as the active, or catalytic site.
The molecule acted on by the enzyme is referred
to as the substrate.
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Evolution of the Heme Group
Molecular oxygen (O2) is only slightly soluble in
aqueous solution. It cannot be carried to tissues
in sufficient quantity if it is simply dissolved
in blood plasma. Diffusion of O2 through the
tissues is also ineffective over distances of
greater than a few millimeters. For these reasons
the evolution of large, multicellular animals
depended on the evolution of proteins that could
transport (e.g., hemoglobin) and store (e.g.,
myoglobin) oxygen. Still, something other than a
protein per se is needed for O2 transport as none
of the 20 standard amino acids can bind oxygen
well. It is thought that this need led to the
evolution of a bound prosthetic group know as
heme which is functional in reversible O2 binding.
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Structure and Properties of the Heme Group
Heme consists of a complex organic ring structure
known as protoporphyrin IX to which is bound a
single iron atom in its ferrous (Fe2) state
(Fig. 5-1). The iron atom has six coordination
bonds, four to nitrogen atoms that are part of
the flat porphyrin ring (Fig. 5-1 d) and two that
are perpendicular to the porphyrin (Fig. 5-1 b,
c, d). The coordinated nitrogen atoms help
prevent conversion of the heme iron to the ferric
(Fe3) state which does not bind O2. Heme is
found in many oxygen-transporting proteins as
well as in proteins such as the cytochromes,
which participate in electron-transfer reactions
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Binding of the Heme Group to Myoglobin
The heme group of myoglobin (and hemoglobin) is
sequestered within a crevice, or pocket, in the
protein (Refer to Fig. 4-16). The burying of the
heme prevents a reaction that would occur with
free heme in solution in which one O2 binds to
two sandwiched heme groups and oxidizes iron to
Fe3. In myoglobin (and hemoglobin), only one of
the perpendicular coordination positions of heme
Fe2 actually is unoccupied and available for O2
binding (Fig. 5-2). The other position is
occupied by a His residue of the polypeptide
chain that is commonly called the proximal
histidine.
When O2 binds to heme the electronic properties
of the iron atom change and solutions containing
the heme turn from a dark purple to a bright red
color. Note that other small molecules such as
carbon monoxide (CO), nitric oxide (NO), and
cyanide ion (CN-) can replace O2 in the open
coordination position. As discussed below,
structural features of the heme binding pocket
interfere with the binding of these other ligands
and reduce their strength of interaction with
heme iron.
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The Globin Family
The globins are a widespread family of proteins
that are commonly found in eukaryotes of all
classes and even in some bacteria. Most globins
function in oxygen transport or storage, although
some are involved in the sensing of gases such as
O2, NO, and CO. At least four types of globins
occur in humans and other mammals. These include
the monomeric myoglobin, which facilitates oxygen
diffusion in muscle tissue, and the tetrameric
hemoglobin, which transports oxygen in the blood.
A third monomeric globin, neuroglobin, is
expressed in neurons and helps protect the brain
from hypoxia (low oxygen) and ischemia
(restricted blood supply). A fourth monomeric
globin, cytoglobin, is found in number of
tissues, but its function is unknown.
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The Structure of Myoglobin
Myoglobin (Mb) is a monomeric, globular protein
(153 aas, Mr 16,700) that contains one heme
prosthetic group. There are eight ?-helical
segments in Mb and other globins, and these are
labeled A through H consecutively in the sequence
(Fig. 5-3). The bends in the molecule are
designated AB, CD, EF, and so forth, based on the
?-helical segments they connect. Globins have an
unusually high percentage of ?-helical structure
(78 in Mb). In our discussion of Mb (and
hemoglobin) structure and function, we will refer
to several residues in the sequence. Note that
these residues can be numbered based on their
positions in the amino acid sequence, or by their
locations in the sequence of a particular
?-helical segment. For example, the His residue
that is coordinated to the heme group of Mb can
be referred to as His93 or His F8.
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Mathematical Description of Protein-ligand
Binding (I)
The reversible binding of a protein (P) to a
ligand (L) can be described by the equilibrium
expression P L ? PL The reaction is
characterized by an equilibrium constant, Ka, Ka
PL/PL ka/kd where ka and kd are rate
constants for association and dissociation,
respectively. Ka is the association constant that
describes the equilibrium between the complex and
the unbound components of the complex. The Ka
provides a measure of the affinity of the ligand
for the protein. Ka has units of M-1, and the
higher the value of the Ka, the higher the
affinity of the ligand for the protein. A
rearrangement of the equilibrium equation shows
that the ratio of bound to free protein is
directly proportional to the concentration of the
free ligand. KaL PL/P
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Mathematical Description of Protein-ligand
Binding (II)

• The binding equilibrium can be described from the
  standpoint of the fraction (theta, ?) of ligand
  binding sites in the protein that are occupied by
  ligand
• binding sites occupied/total binding sites
  PL/(PL P)
• Substituting KaLP for PL and rearranging
  terms gives
• KaLP/(KaLP P) KaL/(KaL 1)
  L/(L 1/Ka)
• The value of Ka can be determined graphically
  from a plot of ? vs the concentration of the free
  ligand, L (Fig. 5-4a, next slide). Note that an
  equation of the form x y/(y z) describes a
  rectangular hyperbola, and ? therefore is a
  hyperbolic function of L. The fraction of
  ligand-binding sites occupied approaches
  saturation asymptotically as L increases. The
  L at which half of the available ligand-binding
  sites are occupied (? 0.5) corresponds to 1/Ka.

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Mathematical Description of Protein-ligand
Binding (III)
In the curve, the L at which half of the
available ligand binding sites are occupied is
equivalent to 1/Ka, or Kd (see next slide). The
curve has a horizontal asymptote at ? 1.0 and a
vertical asymptote (not shown) at L -1/Ka.
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Mathematical Description of Protein-ligand
Binding (IV)

• It is more common (and intuitively simpler), to
  consider the dissociation constant, Kd, which is
  the reciprocal of Ka (Kd 1/Ka) and is given in
  units of molar concentration (M). Kd is the
  equilibrium constant for the release of ligand
  from the complex. Expressions analogous to those
  derived above can be written using the Kd, namely
• Kd PL/PL kd/ka
• and
• L/(L Kd)
• The latter equation plots the curve shown in Fig.
  5-4a in the preceding slide.
• When L equals Kd, half of the ligand-binding
  sites are occupied. As L falls below Kd,
  progressively less of the protein has ligand
  bound to it. In order for 90 of the available
  ligand-binding sites to be occupied, L must be
  9 times greater than the Kd. Finally, note that a
  lower value of the Kd corresponds to a higher
  affinity of the ligand for the protein (see Table
  5-1, next slide).

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Worked Example 5-1. Receptor-ligand Dissociation
Constants
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Analysis of O2 Binding to Mb (I)
A series of equations describing the binding of
O2 to Mb can be derived from the equations used
to describe the general binding of a ligand to a
protein. First, the concentration of dissolved
oxygen can be substituted for L into the
equation relating ? to L and Kd, namely ?
O2/(O2 Kd) As for any ligand, Kd equals the
O2 at which half of the available
ligand-binding sites are occupied, or O20.5. ?
O2/(O2 O20.5) In experiments using
oxygen as a ligand, it is the partial pressure of
oxygen (pO2) in the gas phase above the solution
that is varied. pO2 is easier to measure than the
concentration of O2 dissolved in solution. The
concentration of a volatile substance in solution
is always proportional to the partial pressure of
the gas. So if we define the partial pressure of
oxygen at O20.5 as P50, substitution into the
above equation gives ? pO2/(pO2 P50)
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Analysis of O2 Binding to Mb (II)
A binding curve for Mb that relates ? to pO2 is
shown in Fig. 5-4b. The partial pressure of O2 in
the air above the Mb solution is expressed in
kilopascals (kPa). Oxygen binds tightly to Mb,
with a P50 of only 0.26 kPa.
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Protein Structure Affects Ligand Binding (I)
The specificity with which heme binds its various
ligands is altered when heme is a component of Mb
(and hemoglobin). For example, CO binds to free
heme molecules more than 20,000 times better than
O2 (that is the Kd or P50 for CO binding to free
heme is more than 20,000 times lower than that
for O2). However, CO only binds about 200 times
better than O2 when the heme is bound to Mb. This
difference may be partly explained by steric
hindrance (next slide). When O2 binds to free
heme, the axis of the oxygen molecule is
positioned at an angle to the Fe-O bond (Fig. 5-5
a). In contrast, when CO binds to free heme, the
Fe, C, and O atoms lie in a straight line (Fig.
5-5 b). In both cases, the binding alignment
reflects the geometry of the hybrid orbitals in
each ligand. (Continued on the next slide).
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Protein Structure Affects Ligand Binding (II)
In Mb, His64 (His E7, the distal histidine) on
the O2-binding side of the heme is too far away
to coordinate with the heme iron, but it does
interact with ligands bound to heme (Fig. 5-5 c).
In fact, His E7 may help preclude the linear
binding of CO, providing one explanation for the
selectively diminished binding of CO to heme in
Mb (and hemoglobin). The binding of O2 to heme in
Mb depends on molecular motions, breathing, in
the structure of the polypeptide chain. The heme
group is buried so deeply in folded Mb that there
is no direct path for O2 to the coordination
site. In order for O2 to reach its binding site
in heme, side-chains of Mb must move out of the
way and produce transient cavities in the
structure through which O2 can move.