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NON COVALENT INTERACTIONS
Introduction
The non covalent interactions are of great importance as they define and
stabilize the three-dimensional structure of a protein and its interaction with
other molecular partners. The non-covalent interactions (Fig. 1) are extremely
weak, and contribute to the stabilization of the molecule by a few kcal / mol
and, in some cases, even for a few tenths of kcal / mol. In a macromolecule
weak interactions are so numerous that their contribution is crucial for the
definition of the structure. They are: interactions of Van der Waals,
electrostatic interactions, hydrogen bonds, hydrophobic interactions. The latter
is the tendency of polypeptides to be excluded from interaction with water
molecules, a phenomenon called "hydrophobic effect".
Figura 1. List of non covalent interactions
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Van der Waals interactions
Each pair of atoms has an optimal distance. When the atoms are too close, the
orbit of the outer electrons tend to overlap and they repel each other, the
repulsion increases with decreasing the distance, below a certain threshold
distance there is a real barrier, this distance defines the Van der Waals radius
(Fig. 2). Each atom has its own inviolable space, it follows that the Van der
Waals radius set the limits of the compactness of the structure.
Figura 2. Van der Waals radius of the atoms of a water molecule.
Electrostatic Interactions
The electrostatic interactions can be due to the interactions of monopoles
1
(single
charges),
or
of
dipoles
1
Electric dipole: system made by 2 identical electric charges of opposte sign, separated by a distance d.
The absolute value of one of the two charges multiplied by the distance gives the value of the dipole
moment that it is expressed in Debye
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and then due to opposite charges separated by a definit distance.
The hydrogen bond occurs between a donor and an acceptor of hydrogen
atoms. When the interaction takes place between charged groups it is often
referred to as salt bridge and it has properties typical either of an electrostatic
interaction either of an hydrogen bond.
The weak bonds between atoms with opposite charges are very important
because in a protein there are many charged amino acids. The primary role of
the charges is to make the protein soluble in an aqueous solvent, they also play
a vital role in the stability of the macromolecule and in the detection of
molecular partners, such as in the enzyme-substrate recognition or in the
protein-protein interactions through the formation of specific salt bridges.
The potential around a protein can be measured by the Coulomb law. Once
known the charges distribution on the protein and their relative distance it is
possible to calculate the electrostatic potential with the formula represented in
Figure 3, where ε is the dielectric constant, q1 and q2, the value of electric
charges, R the distance, ΔE the potential acting between the 2 charges. A
contribution to the elecrostatic potential is also given by the electric dipoles,
consisting of two opposite charges separated by a distance d.
Figura 3. The Coulombs law and an
electric dipole
The dipole-dipole interaction depends on the orientation of a dipole over the
other (parallel, linear, opposite) and is maximum when the two dipoles are
linear or opposed. The analysis of a molecule such as HCl (Fig. 4) allows us to
understand what is a dipole moment. The distance between the H and Cl is 1.3
Å, if the charges were at this distance, the value of the dipole moment would
be 6 Debye, as the value of the dipole moment is given by the absolute value
of the charge multiplied by the distance. The experimentally measured dipole
moment of the HCl molecule is about 1 Debye, which means that the charge
delocalization is about 17% of the total charge. Two electrons are shared in the
molecule of HCl, but the chlorine atom exerts an higher attraction on the
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electrons bond, making the chlorine more negative with the consequent
formation of a dipole. The greatest the charge delocalization, the greatest will
be the electric dipole.
Figura 4. Dipole moment of the HCl
molecule.
In a protein a dipole moment is associated to each peptide bond. The dipole
moment of a peptide bond is about 3.5 Debye. The final values depends on the
orientation of the individual dipoles.
In an α-helix the dipole moments of the various peptide bonds have the same
orientation (Fig. 5).
Figura 5. Schema del momento di dipolo del legame peptidico in un’α elica.
It follows a strengthening of the various dipole moments and of their final
contribution. As a consequence a strong delocalization of positive and
negative charge is observed at the N and C-terminal of the helix respectively.
In the proximity of the N terminal region of α-helices are often present
negatively charged groups that provide a stabilization because of electrostatic
interactions. The negative groups can be amino acids such as glutamic and
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aspartic residues, or external groups such as a phosphate group. The
preference for an α helix to have a negatively charged group near the Nterminus has been verified experimentally by measuring the increase in
stability achieved by the introduction of a group with a negative charge by site
directed mutagenesis.
In Table I some characteristics of the amino acids are reported. Here we are
mainly interested to the the pK of the various groups, as it determines the
state of protonation and deprotonation of the lateral chains. At neutral pH the
aspartic and glutamic are normally negatively charged, while the lysine and
arginine are positively charged, in the case of histidine its protonation state
will depend on its microenvironment because its pK in normal condition is
near the neutrality. The pK of the same amino acid changes depending on its
location, the presence of a charge for example, is able to perturb the pK of
other groups. The electrostatic interaction depends on the distance, decreasing
with increasing distance.
Table I. Phsical-chemical properties of the aminoacids
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Figura 6: pK of the N e C-terminal groups of a single alanine or of an alanine forming a
dipeptide, a tripeptide or a tetrapeptide.
The phenomenon is clearly described in the example in Figure 6, where it is
represented an alanine alone or in the form of a dipeptide (alanine 2),
tripeptide (3 alanine) or tetrapeptide (4 alanine). For each of these situations
the pK relative to the protonation / deprotonation equilibrium of the C-and Nterminal can be experimentally measured in solution.
The pK of the carboxylic group in the single-alanine is 2.3 and of the amminic
group group is 9.6, for the dipeptide the pK of the carboxylic group is 3.1 and
of the amminic group becomes 8.3, for the tripeptide 3.4 and 8.0 while for the
tetrapeptide there is no change and the pK values are yet 3.4 and 8.0
respectively. This suggests that, proceeding from a situation with three
alanines to the one with four alanines, the pK values do not change, while
there is a variation of more than one unit of pH going from a system
characterized by a single alanine to the one made by three alanine. By
reducing the number of alanines the pK value of the carboxylic group
decreases. In summary, it is more difficult to protonate the carboxylic group of
single alanine than that of a tetra-alanine. The carboxyl, in fact, prefers to stay
deprotonated with a negatively charge, in order to have a stabilizing
interaction with the positive charge of the N-terminal group and a lower pH
must be reached to obtain its protonation. In the case of the single alanine the
carboxy-terminal and the ammino-terminal are much closer than in the case of
a tri-alanine because the distance between the two groups increases with the
number of alanine. The pK of a chemical group is strongly influenced by the
presence of another charge, therefore it is necessary to know the distribution of
charges in a protein to determine the pK of the charge of interest. The
electrostatic interaction depends on the distance between the different charges
and this example demonstrates how the pK value is affected by the presence of
a charge. The introduction or removal of new charges by site directed
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mutagenesis is a strategy used to modulate a chemical environment and
therefore influence the state of protonation of the groups close to them.
In this example, the unperturbed system is constituted by a tetra-alanina
because the C-terminal group does not feel the presence of N-terminal group,
while the perturbed system is constitted by the mono-alanine because the N
and C-terminal are close enough to influence their pK value. It is possible to
measure the energy associated to the pK variation due to the presence of a
charge. Considering the value of pK of the carboxylic group in the
unperturbed (3.4) and the perturbed (2.3) system, the difference of free energy
coupled with this change, that is equal to 2.5 kcal / mol, can be calculated
(Fig. 7). Then the energies involved in electrostatic interactions, (and this will
be true for all other weak interactions) are of the order of a few kcal / mol. In
the case of carboxylic acid group, in the perturbed situation the group tends to
dissociate more easily and to be protonated with more difficulty.
Figura 7. Free energy differente
associated to the pK variation.
When we refer to the pK of a group in a protein, we refer to its apparent pK
value. This is not the actual pK of the group, but the value that shows the
group in a given chemical environment. Approximating a globular protein
with a sphere, the majority of charges will be distributed on the surface of the
sphere and only a few will be in the internal region. It is possible to calculate
the value of the electrostatic potential around a macromolecule by applying the
Coulomb's law.The calculation may be more accurate by solving the PoissonBoltzmann equation and considering the presence of two dielectrics, a region
with a high value (80), formed by water and the other with a low value (3-4),
consisting of the protein. The potential is usually calculated at neutral pH,
39
determining the contribution of individual groups with their state of
protonation and is represented drawing equipotential lines, i.e. lines in which
the potential has the same value. An example is shown in Figure 8, which
represents the distribution of the potential of copper and zinc superoxide
dismutase (SOD). In this representation it can be seen as an external molecule
perceives the macromolecule from an electrostatic point of view.
Figura 8. equipotential lines around the Cu,Zn superoxide dismutase
The knoweledge of the distribution of the equipotential lines can be crucial to
understanding the mechanisms of protein-protein or enzyme-substrate
recognition. In the case of superoxide dismutase, for example, the substrate
superoxide is a negatively charged molecule, that can interact with the enzyme
only through the lines indicating the presence of a positive potential ..
The hydrophobic effect
The hydrophobic effect arises from the fact that biological macromolecules are
in an aqueous solvent and water did not favor an interaction with non-polar
atoms. This effect has a dominant role in the stability of biological
macromolecules and has some unusual properties.
40
In figure 10 we summarized a number of peculiarities of the water that is one
of the few liquids that expands when it freezes.
Figura 10. Hydrogen bonds in the ice
When ice melts it begins to shrink and this phenomenon persists up to a
temperature of 4 ° C, after this temperature the thermal agitation
counterbalances the phenomenon of contraction. The phenomenon of
expansion due to freezing is because the ice is composed of water molecules
highly ordered, which increase their distance to optimize their hydrogen
bonds.
Another important property of water is to have a dipole moment of the value
of 1.8 Debye, which allows it to be either a good acceptor or a good donor of
hydrogen bond. The presence of hydrogen bond features a variety of
properties of water, both at the microscopic and macroscopic level. The
relevance of hydrogen bond is highlighted in Table III that shows the melting
and boiling point of the H2O and H2S molecule.
Tabella III. Comparison of the melting and boiling point of molecules of similar size
41
The value is very different and the reason is that the water molecules form
hydrogen bonds and therefore require a greater temperature, or a greater
amount of energy, to separate the molecules and have then higher melting and
boiling temperature. The water forms ideal hydrogen bonds in the ice state
where the water molecules are strongly ordered and a partial order is also
maintained in solution. This implies that water molecules are partially ordered
in the liquid phase.
The radial distribution function of water is reported in figure 11 is. The plot
indicates the probability of finding a molecule at a certain distance from
another and then yet another and so on. The figure shows that there is a peak at
around 3 Å and then another one at around 4 / 5 Å. At the top of the figure,
where the distance is expressed in terms of the diameter of the van der Waals
molecule, it is possible to observe the presence of a peak for each multiple of
the diameter, with decreasing intensity.
Figura 11. Radial distribution function of the
water molecule
The presence of water molecules well arranged in the first coordination sphere
but also in the second and in part in the third sphere, denotes the ability of
water to create a network of hydrogen bonds in solution, although not so
perfect and defined as in the case of ice. This arrangement is important from
the viewpoint of solubility and determines the ability of molecules or
macromolecules to associate or not with each other. For the understanding of
the phenomenon it is necessary to evaluate the total energy of the system.
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Table IV. Association constant for different kind of molecules in water.
Table IV shows the association constant for a number of small molecules, i.e.
their capacity to associate one with the other when placed in an aqueous
solvent. The association constant (which is measured in M-1) is relatively high
for molecules that are able to make salt bridges and is of the same order of
magnitude for molecules that are hydrophobic, thus able to interact with each
other because of the hydrophobic effect , while it is lower (with a differente of
a factor of 10 ) for polar molecules that can interact with each other only
through hydrogen bonds. Water is able to modulate the ability of association
of the molecules according to their properties.
In detail: small molecules that can interact with each other as well as with the
water molecule will have an association constant of 1 / 55 = 0.02 M-1,
because 55 M is the concentration of water in liquid phase. The association
constant indicates the tendency of two molecules to stay together and is given
by:
KAB= [AB] / [A] [ B] M-1
Two molecole, in order to associate one with another, must overcome an
entropic barrier, reducing its degree of freedom, and need a more favourable
energetics in the interaction between them than between each molecule and
water. The association constant between two molecules is strongly dependent
on the solvent. Figure 12 describes the behavior in different solvents of the
metilacetamide molecule, used as a model compound for the peptide bond,
because an hydrogen bond can be establish between the CO and NH of the
methylacetamide molecule.
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Figura 12. Formation of the mathylacetamide dimer in different solvents
Figure 12 shows the percentage of the dimeric form of N-methylacetamide as
a function of its concentration. The dimeric form exists due to the possibility
to form a hydrogen bond between the CO and NH group of two molecules. In
a solvent such as CCl4, already at low concentration, the N-methylacetamide
is mainly in the dimeric form, whereas in a solvent such as water it is
necessary to increase the concentration up to 10 M. This is due to the
competition that occurs between water and the same molecule in the formation
of a hydrogen bond, which prevents the methylacetamide to take the dimeric
form. The ability to form an hydrogen bond is monitored by infrared
spectroscopy, observing the vibration band of the NH group, that is different
depending on whether the molecule is in the monomeric or dimeric form (the
proton, interacting with the carbonyl of another molecule, vibrates differently).
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Tabella V. Free energy variation for the transfer of various molecules from ethanol to
water at 25 °C.
The previous example shows how the solvent significantly modulates the
properties of the solutes dissolved in the solvent itself, and this happens also
when the solutes are amino acids. In particular, each amino acid has specific
hydrophobic or hydrophilic characteristics, determined by the chemical
composition of its side chain. In Table V is reproduced the change of free
energy (ΔG) for the transfer of a definite molecule from ethanol to water. The
ΔG of transfer evaluates the propensity of a molecule to be dissolved in an
aqueous or in a more hydrophobic solvent such as ethanol. The measurement
of the ΔG of transfer for all amino acids permits to obtain a scale on the
hydrophobic or hydrophilic characteristics of each amino acid. The ΔG of
transfer may be measured by assessing the solubility of the molecule in water
and ethanol and by calculating the logarithm of the ratio of the two solubility.
A value of ΔG <0 indicates a hydrophilic molecule, while a value of ΔG> 0
indicates a hydrophobic molecule. Since each amino acid has a carboxylic and
an amminic group, the ΔG of transfer from ethanol to water is negative for
each aminoaid because the negative and positive charge tend to push it toward
the aqueous solvent. You can get the ΔG of each side chain taking the glycine
as a reference. In fact glycine has not a side chain, and subtracting each time
its value it is possibile to assess the hydrophobic or hydrophilic characteristics
of each side chain and build up a relative scale of hydropatie. It is possibile to
do the same for other molecules such as ethane and methane. The values
reported in the table indicate that any chemical group gives an equal
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contribution regardless of the molecule it belongs to. In the case of CH2, for
example, this contribution is the same if you consider it as the result of the
subtraction from an ethane and methane molecule, or from an alanine and a
glycine or from a leucine and valine. It follows that the CH2 group gives a
contribution of 0.7 kcal / mol regardless of the molecule where it is found.
If you calculate the ΔG transfer of methane from benzene (as hydrophobic
solvent) to water ( fig.13), the value is positive since the methane does not like
a hydrophilic environment. In figure 13 are reported the enthalpic and entropic
contribution related to the transfer.
Figura 13. Free energy variation
from
the
transfer
from
hydrophobic
to
hydrofilic
solvents.
Figura 14. Scheme of chlathrate to dissolve an
hydrophobic molecule
The tendency of hydrophobic molecules to not be transferred to the
hydrophilic environment is mainly due to an entropic reason. ΔH is in fact
negative, indicating that from the entalpic point of view the molecule likes to
be dissolved in water, but the resulting ΔS is strongly negative and since ΔG =
ΔH-TΔS, the final ΔG value is positive. Figure 14 represents a hydrophobic
molecule dissolved in aqueous environment: the water molecules form an
ordered cage or chlathrate building a cavity where the hydrophobic molecule
can insert itself. The water molecules are highly ordered providing an
explanation for the strong entropic cost related to the hydrophobic effect.
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Figura 15. Transfer for amino acid lateral
chain from water to ethanol
This effect increases as the solvent accessibile surface of the molecule
increases. The graph of the ΔG of transfer from water to ethanol for the side
chain of a non polar amino acid is reported in Fig. 15 as a function of the area
accessible to solvent (SAS). The trend is linear, increasing the size of the side
chain the ΔG values increase and are more negative since a larger cavity in the
water solvent must be built to accomodate the hydrophobic molecule. The
value of the slope of this line corresponds to 20 cal / mol A2. This means that
each A2 of the surface gives a contribution of 20 cal / mol relative to the
hydrophobic interaction, a value that corresponds to the value obtained from
experiments on the stability of protein macromolecules
For example, in the case of lysozyme the change of stability has been
evaluated by following a series of mutations of hydrophobic amino acid
located in the interior of the protein (Fig.
16).
Figura 16. Effect on the stability of a protein due
to the formation of internal cavities
In detail leucines located in to the protein hydrophobic core have been mutated
in alanines, to create cavities. The effect on the protein stability has been
experimentally evaluated measuring the ΔG of denaturation for the native and
the mutated form. The destabilization is directly proportional to the cavity
created in the protein (Fig. 16), and the slope of the straight line has a value of
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about 20 cal / mol A2. It is confirmed that an area of about 100 A2 offer a
contribution to the stabilization of the order of 1-2 kcal / mol.
In summary, when a hydrophobic solute is dissolved in an aqueous solvent
three events occur that determine the total energy of the process:
1. creation of a cavity in the solvent,
2. introduction of the solute in the cavity
3.
rearrangement of the solute and solvent in order to optimize the
interaction.
An hydropatic relative scale for the amino acids can be used to predict
segments of a protein that are located within a membrane. In fact, once
determined the values of ΔG transfer for all side chains, you can define a scale
that define for instance how arginine is more hydrophilic than glycine or
leucine. In this way you can predict the degree of hydrophobic character of a
segment of a protein and identify segments which have a good chance of
being inside of a double lipid layer. To this end sequences of segments of
length between 17 and 21 amino acids (corresponding to the number of
residues needed to cross from side to side in a membrane conformation to αhelix) are analyzed. The analysis performed using for example windows of 17
residues permits to construct an hydropatic graph that evidentiate the degree of
hydrophobicity of segments of this length as a function of the amino acidic
sequence. In practice for the first 17 residues , the average hydrophobic value
is calculated and is reported in the plot on the middle of the segment.
The same procedure is applied to the segment that runs from residue 2-18, and
to the subsequent segments (Fig. 18).
Figura
18.
Prediction
of
transmembrane through the use
of an hydropatic plot.
Figure 19 represents the predictive analysis of the transmembrane segments of
the M and L subunit of the photosynthetic reaction center. The graph shows
that there are segments that have a strong hydrophobic connotation,
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corresponding to segments that have a high probability of being inside the
membrane..
Figura 19. Prediction of trans
membrane segments of the M and L
subunits of the photosynthetic
reaction center.
It should be stressed that the transmembrane segments, although having a
hydrophobic character, may also contain charged aminoacids which are often
used to interact with its specific substrate..
Hydrogen bond
The other weak interaction of great importance is the hydrogen bond that has
an important role in modulating the stability of a biological macromolecule,
but that is also crucial in the processes of macromolecular recognition.
The hydrogen bond is a polar interaction in which one atom of hydrogen is
partially shared by two electronegatives atoms. The hydrogen can be
considered as a proton that is partially dissociated from a donor atom, thus
allowing the sharing by an acceptor atom. The presence of an hydrogen bond
can be defined simply by geometric criteria. So there is a hydrogen bond if the
distance between the donor and acceptor atom is 3 Ǻ and the angle between
the donor, the hydrogen and the acceptor is equal to 180 ± 60 ° (Fig. 20).
When the angle is 180 °, all three atoms are aligned, so the hydrogen bond
interaction is optimal..
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Figura 20. Scheme of an hydrogen bond
The angle can vary, but if it becomes less than 120 ° it is not possibile any
more to share the hydrogen atom between the donor and the acceptor (Fig. 21).
Figura 21. Two hydrogen
bond
with
different
geometric parameters.
The hydrogen bond in a protein can be formed between several chemical
groups of the side chain, but often occurs at the level of the main chain,
between the carbonyl and the amide and is often correlated with the formation
of the preferential secondary structures. The macromolecule is capable of
forming hydrogen bonds also with the solvent molecules and the external
substrate and the occurrence of a selected preferential interaction can be
evaluated considering the total energetics of the system.
In cases where there are, for example, two molecules that form a hydrogen
bond between themselves and with the solvent, it is necessary to determine the
energy of interaction before and after the intermolecular interaction has taken
place. The two molecules may be an enzyme and a substrate (SB). The
enzyme and the substrate in solution, before interacting one with the other,
will have a number of groups able to interact with water through hydrogen
bonds. The enzyme-substrate interaction allows the formation of one or more
hydrogen bonds between the enzyme and the substrate, following the
displacement of water molecules that will then form a hydrogen between
themselves. If the formation of intermolecular bond is favorable the reaction
is directed toward the formation of the enzyme-substrate bond. To understand
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the direction of the reaction the total energetics of the system must be
evaluated, calculating the total number of hydrogen bonds before and after the
reaction to assess the direction of the reaction from the enthalpic viewpoint.
Similarly we should proceed from the entropic viewpoint. In summary, rather
than determine the absolute value of the hydrogen bond, the energetics must
be assessed before and after the enzyme-substrate bond formation, (ie the
number and type of interactions existing), evaluating if it is more favourable
for the enzyme and the substrate to interact with each other or with the solvent.
Evaluation of the hydrogen bond energetic contribute
A simple method to assess the effect of a hydrogen bond in determining the
binding of a ligand (L) to a protein (P), is to compare the protein-ligand
dissociation constant, in two different conditions, i.e for the native protein and
for a mutant after removal of a single residue, that participates in the native
protein to the interaction with the ligand via a single hydrogen bond. The
mutation perturbs the dissociation constant and, through a comparative
analysis, you can identify the contribution made by the specific hydrogen bond
to bind a specific ligand. The dissociation constant KD is given by: [P] [L] /
[PL] and can also be expressed as the ratio of the rate of dissociation k-1 and
the rate of association k1. The relationship between the two rates identifies the
dissociation constant KD which is in turn related to the association constant
KA by the relation KD = 1/KA.
The free energy variation, ΔG, between the free and bound form that
determines the ability of the native protein to bind a specific ligand, is related
to the association constant through the relation ΔG = -RTlnKA or ΔG =
RTlnKD.
KA can be experimentally measured in native and mutated conditions
providing the opportunity to determine the difference in free energy associated
with the binding process, due to the mutation. This difference
ΔΔG = RT (logKD prot nat/KD prot mut)
allows to evaluate how the introduced mutation increases or reduces the ability
of the protein to bind the ligand. If ΔΔG is negative then the mutant has a
reduced capacity to bind the ligand compared to the native protein. The
reduced capacity can be attributed to the elimination of the single hydrogen
bond, thereby outlining its contribution in identifying a molecular partner
(which in this case is a ligand). Conceptually, the experiment is very simple:
the protein recognizes the ligand through a series of interactions, including a
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hydrogen bond, which is eliminated by site directed mutagenesis. The value of
the dissociation constant before and after mutation evaluate the weight of the
individual hydrogen bond on the binding efficiency.
A measure on the thermodynamic contribution of a single hydrogen bond can
also be determined using, as an indicator, the parameters that define the
enzymatic kinetics as kcat, KM and kcat / KM. In the following diagram the
general structure of an enzyme reaction is depicted with E, S and P
representing the enzyme, substrate and product, while k1 k-1 and k2 identify
the rate constants associated with the different steps of the mechanism
k2
k1
E + S  ES  E + P
k 1
The contribution that the functional groups provide to an enzyme catalysis can
be estimated by comparing thesteady-state parameters of the kinetics for the
native and the mutant enzyme. Recall that kcat / KM can be regarded as a
second order rate constant. This implies that can be used to describe the
process that proceeds from reactants to the transition state. The transition state
is the state with the highest energy in the scheme of the reaction coordinate
The measurements of kcat and KM for the native enzyme and a series of
mutants, can be used to examine the role of a single side chain binding the
substrate via an hydrogen bond. From the measurement of the enzyme kinetic
constants it is possibile to derive the energetics of the single hydrogen bond
that has been deleted. In summary, thermodynamic parameters are calculated
measuring parameters typical for the enzymatic kinetics. The enzyme that is
taken into account as an example is the tyrosil-tRNA-synthase, which
catalyzes the aminoacilation of tRNA with tyrosine. The reaction occurs in
two steps: the first one consists in the activation of tyrosine to form the tyrosil
adenylate with the release of pyrophosphate, the second one concerns the
transfer of tyrosine to tRNA with the release of AMP (Fig. 23)
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Figura 23. Reaction scheme of the tirosil-tRNA synthase.
The intermediate product is unstable, but incubation of the enzyme with ATP
in the presence of pyrophosphatase, that hydrolyses the pyrophosphate,
prevents the reverse reaction, making the tyrosil adenylate, linked to the
enzyme, a stable intermediate that can be crystallized, allowing to describe the
interaction at the atomic level. The tyrosil adenylate creates a network of 11
hydrogen bonds with the native enzyme (Fig. 24). The hydrogen bonds are
selectively and individually removed and the effect is verified by measuring
the enzyme efficiency. In detail comparison of the kcat / KM ratio of the
native and mutated protein can be used to evaluate the energetic contribution
of the eliminated hydrogen bond. Figure 24 shows the presence of the
extensive network of hydrogen bonds that occur between charged and polar
groups and it is conceivable that the contribution of the hydrogen bond can be
different depending on whether it is made by charged or polar groups.
Figura 24. Hydrogen bonds network between the enzyme and the tyrosiladenilate.
We now analyze the effect of specific mutations. Mutations of tyrosine →
phenylalanine-34 and cysteine → glycine-35 concern the elimination of polar
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groups of the protein that make hydrogen bond with other polar groups of the
substrate.
Tabella VI. Kinetics parameters of the tyrosil-tRNA sinthase.
The cysteine-glycine mutation leads to a change in kcat / KM of about a factor
of 3-4 from 3.7 × 106 to 1.1 × 106. The change is relatively small and the
energetic contribution, due to the single hydrogen bond is around 1 kcal / mol
(Table VI). The tyrosine-phenylalanine mutation leads to a change in the kcat /
KM of about a factor of 2-3 from 3.7 × 106 to 1.5 × 106 and the relative
energetic contribution is around 0.5 kcal / mol. The variation of the value of
kcat / KM is then correlated to the loss of the hydrogen bond that occurs for
example between cysteine and the hydroxyl group of ribose.
The mutation of histidine 48 in glycine leads to the loss of a hydrogen bond
that occurs between a positively charged residue (histidine 48) and a polar
atom of ribose. In this case the effect of mutations should lead to a greater
change in energy since the hydrogen bond between a positively charged and a
polar group should be stronger than that between two polar groups. However,
Table VI shows that the change of kcat / KM due to this mutation, is about a
factor 6 and the corresponding free energy change is approximately 1 kcal /
mol, ie the same value of the previous mutations.
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This result can be understood analyzing the energetics of the enzyme and the
substrate before and after their interaction as shown in Figure 25.
a)
b)
c)
d)
Figura 25. Scheme of the hydrogen bond between the enzyme and the substrate
In the first case a polar residue (cysteine 35), that forms a hydrogen bond with
a polar group of the substrate, has been mutated. In the native state, before the
enzyme-substrate interaction, the enzyme and in particular cysteine 35 forms a
hydrogen bond with a water molecule and the same occurs for the substrate
(a). When the enzyme and the substrate interact, they form a hydrogen bond
and and an ydrogen bond i salso formed by the relesead water molecules. The
mutation of the cysteine produces the removal of a single hydrogen bond from
the enzyme both on the right and the left side of the reaction, due to the
interaction of cysteine with the substrate or with a water molecule respetively,
and a single hydrogen bond between the substrate and a water molecule and
between water molecules will be maintained , so that the reaction will be
isoentalpic ( b).
In presence of a charge such as an histidine, before the recognition between
the enzyme and the substrate the charge of the histidine forms a charge-dipole
hydrogen bond with the water molecule, that is more stable than a dipoledipole hydrogen bond. It follows that to the left of the reaction, a chargedipole hydrogen bond as well as a dipole-dipole hydrogen bond between the
substrate and water are present (c), but a similar situation occurs to the right of
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the reaction, where there is either a charge-dipole hydrogen bond (between the
enzyme and substrate) and a dipole-dipole hydrogen bond between the
molecules of water (c). The histidine mutation causes the deletion of the
charge-dipole hydrogen bond, but this occurs both on the right and left side of
the reaction (d), so that the reaction is still isoentalpic. This explains why the
elimination of the histidine (a charged residue) results in a decrease to the
contribution of the substrate binding identical to the elimination of a non
charged residue. In both case the energy variation ΔG is of the order of 1 kcal /
mol.
The effect is different when tyrosine 169 is mutated in phenylalanine. In the
native proteine tyrosine 169 forms a hydrogen bond with a positively charged
group of the substrate. The effect of the mutation on the specificity constant
kcat / KM is large, because the value goes from 106 to 103 and the change in
energy is of the order of 4 kcal / mol (Table VI). Therefore, the mutation of a
residue that forms a hydrogen bond with a charge of the substrate, results in a
reduction of the specificity of a thousand, from 106 to 103 and a change in the
energy of 4 kcal / mol. The motivation is clear from the analysis of the
energetics of the reaction (Fig. 26)
Figura 26. Scheme of the reaction due to the deletion of a residue interacting with a
charged group of the substrate
Before the enzyme-substrate interaction, the native enzyme interacts with the
water molecule through a dipole-dipole interaction, while the substrate
interacts with the water through a charge-dipole interaction. After the enzymesubstrate recognition we have again a charge-dipole interaction ( between the
enzyme and the substrate) and a dipole-dipole interaction (between the
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molecules of water). The mutation of the enzyme residue that interacts with a
charge of the substrate results in an asymmetric balance. On the left ther is a
charge-dipole hydrogen bond but on the right there is only a dipole-dipole
hydrogen bond, the reaction is then pushed toword the left side. The reaction is
not any more isoentalpic and the energy variation due to the loss of this
hydrogen bond is 4 kcal / mol, with a consequent significant varietion of the
enzyme specificity constant kcat / KM, that is reduced by a factor of thousand.
The example confirms that the hydrogen bonds provide energy contributions
of a some kcal / mol, but also demonstrates their importance in terms of
recognition and specificity, especially when involving charged groups. The
analysis of the energetico of the system is essential to understand that the
effect depends on the position of the charge if the charge is placed on the
protein (eg. the histidine 48) the loss of specificity is very low, but if the
charge is placed on the substrate is large, because the reaction is not any more
isoentalpic. It should be stressed the importance of hydrogen bonds in the
processes of recognition. The mutation of tyrosine 69 in phenylalanine
determines a reduced specificity for the substrate by a factor of 1000, so the
ability of the enzyme to recognize and react with the defined the substrate is
1000 times decreased.
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