CHAPTER 4
Biophysical Principles
T he concepts in this chapter form the basis for under-
standing all the molecular interactions in chemistry and
biology. To illustrate some of these concepts with a
practical example, the chapter concludes with a section
on the exceptionally important Ras family of enzymes
that bind and hydrolyze the nucleotide guanosine tri-
phosphate (GTP). This example provides the background
knowledge to understand how guanosine triphospha-
tases (GTPases) participate in numerous processes
covered in later chapters.
Most molecular interactions in cells are driven by dif-
fusion of reactants that simply collide with each other
on a random basis. Similarly, dissociation of molecular
complexes is a random process that occurs with a prob-
ability determined by the strength of the chemical bonds
holding the molecules together. Many other reactions
occur within molecules or molecular complexes. The
aim of biophysical chemistry is to explain life processes
in terms of such molecular interactions.
The extent of a chemical reaction is characterized by
the equilibrium constant; the rates of the reactions are
described by rate constants. This chapter reviews the
physical basis for rate constants and how they are related
to the thermodynamic parameter, the equilibrium con-
stant. These simple but powerful principles permit a
deeper appreciation of molecular interactions in cells.
On the basis of many examples presented in this book,
it will become clear to the reader that rate constants are
at least as important as equilibrium constants because
the rates of reactions govern the dynamics of the cell
and many processes are controlled at rate-limiting steps.
The chapter includes discussion of the chemical bonds
important in biochemistry.
This chapter is adapted in part from Wachsstock. DH, Pollard TD.
Transient state kinetics tutorial using KINSIM. Biophys J.
1994;67:12601273.
First-Order Reactions
First-order reactions have one reactant (R) and produce
one or more products (P). The general case is simply
RP
Some common examples of first-order reactions (Fig.
4.1) include conformational changes, such as a change
in shape of protein A to shape A*:
A A*
and the dissociation of complexes, such as
AB A + B
The rate of a first-order reaction is directly propor-
tional to the concentration of the reactant (R, A, or AB
in these examples). The rate of a first-order reaction,
expressed as a differential equation (rate of change of
reactant or product as a function of time [t]), is simply
the concentration of the reactant times a constant, the
rate constant k, with units of s1 (per second):
Rate = d[R ] dt = d[P] dt = k[R ]
The rate of the reaction has units of M s1, where M is
moles per liter and s is seconds (molar per second). As
the reactant is depleted, the rate slows proportionally.
A first-order rate constant can be viewed as a prob-
ability per unit of time. For a conformational change, it
is the probability that any A will change to A* in a unit
of time. For dissociation of complex AB, the first-order
rate constant is determined by the strength of the bonds
holding the complex together. This dissociation rate
constant can be viewed as the probability that the
complex will fall apart in a unit of time. The probability
of the conformational change of any particular A to A*
or of the dissociation of any particular AB is independent
of its concentration. The concentrations of A and AB
53
54 SECTION II n Chemical and Physical Background

BOX 4.1 Relationship of the Half-Time to

A A* a First-Order Rate Constant
Conformational
change In thinking about a first-order reaction, it is useful to refer
to the half-time of the reaction. The half-time, t1/2, is the
time for half of the existing reactant to be converted to
A B A + B product. For a first-order reaction, this time depends only
Dissociation on the rate constant and therefore is the same regardless
of the starting concentration of the reactant. The relation-
FIGURE 4.1 FIRST-ORDER REACTIONS. In first-order reactions, ship is derived as follows:
a single reactant undergoes a change. In these examples, molecule A d[ R] dt = k[ R]
changes conformation to A* and the bimolecular complex AB dissoci-
ates to A and B. The rate constant for a first-order reaction (arrows) so
is a simple probability.
d[ R] [ R] = kdt
Integrating and rearranging, we have
are important only in determining the rate of the reac-
tion observed in a bulk sample (Box 4.1). ln[ Rt ] = ln[ Ro ] kt
To review, the rate of a first-order reaction is simply or
the product of a constant that is characteristic of the
[ Rt ] = [ Ro ]e kt
reaction and the concentration of the single reactant.
The constant can be calculated from the half-time of a where Ro is the initial concentration and Rt is the concen-
reaction (see Box 4.1). tration at time t.
When the concentration at the initial time point Ro is
reduced by half,
Second-Order Reactions
1
2 [ Ro ] = [ Ro ]e kt 12

Second-order reactions have two reactants (Fig. 4.2). The

or
general case is
R1 + R 2 product
1
2 = e kt
12

and
A common example in biology is a bimolecular associa-
tion reaction, such as 2 = e kt 12

A + B AB Thus,

where A and B are two molecules that bind together. ln 2 = kt1 2

Some examples are binding of substrates to enzymes,
binding of ligands to receptors, and binding of proteins
to other proteins or nucleic acids.
The rate of a second-order reaction is the product
of the concentrations of the two reactants, R1 and R2,
and the second-order rate constant, k:
Reaction rate = d[P] dt = k[R1 ][R 2 ]
The second-order rate constant, k, has units of M1s1
(per molar per second). The units for the reaction
rate are
[R1 ] [R 2 ] k = M M M 1s 1 or M s 1
the same as a first-order reaction.
The value of a second-order association rate con-
stant, k+, is determined mainly by the rate at which the
molecules collide. This collision rate depends on the rate
of diffusion of the molecules (Fig. 4.2), which is deter-
mined by the size and shape of the molecule, the viscos-
ity of the medium, and the temperature. These factors
are summarized in a parameter called the diffusion
coefficient, D, with units of m2s1. D is a measure of
so, rearranging, we have
t1 2 = 0.693 k
and
k = 0.693 t1 2
Therefore dividing 0.693 by the half-time gives the first-
order rate constant and dividing 0.693 by the rate constant
gives the half-time. This relationship is independent of the
extent of the reaction at the outset of the observations and
allows one to estimate the rate constant without knowing
absolute concentrations.
how fast a molecule moves in a given medium. The rate
constant for collisions is described by the Debye-
Smoluchowski equation, a relationship that depends
only on the diffusion coefficients and the area of interac-
tion between the molecules:
k = 4 b( DA + DB ) N o103
where b is the interaction radius of the two particles (in
meters), the Ds are the diffusion coefficients of the

+ the reaction and the concentrations of the two reactants.
A B A B
Slower Slower
C + D C D
Slower Faster
D + D D D
Faster Faster
FIGURE 4.2 SECOND-ORDER REACTIONS. In second-order
reactions, two molecules must collide with each other. The rate of
these collisions is determined by their concentrations and by a collision
rate constant (arrows). The collision rate constant depends on the sum
of the diffusion coefficients of the reactants and the size of their interac-
tion sites. The rate of diffusion in a given medium depends on the size
and shape of the molecule. Large molecules, such as proteins, move
more slowly than small molecules, such as adenosine triphosphate
(ATP). A protein with a diffusion coefficient of 1011m2s1 diffuses
about 10m in a second in water, whereas a small molecule such as
ATP diffuses 100 times faster. The rate constants (arrows) are about
the same for A + B and C + D because the large diffusion coefficient
of D offsets the small size of its interaction site on C. Despite the small
interaction size, D + D is faster because both reactants diffuse rapidly.
reactants, and No is Avogadros number. The factor of
103 converts the value into units of M1s1.
For particles the size of proteins, D is approximately
1011m2s1 and b is approximately 2 109m, so the
rate constants for collisions of two proteins are in the
range of 3 108M1s1. For small molecules such as
sugars, D is approximately 109m2s1 and b is approxi-
mately 109m, so the rate constants for collisions of a
protein and a small molecule are approximately 20 times
larger than collisions of two proteins, in the range of
7 109M1s1. On the other hand, experimentally
observed rate constants for the association of proteins
are 20 to 1000 times smaller than the collision rate con-
stant, on the order of 106 to 107M1s1. The difference
is attributed to a steric factor that accounts for the fact
that macromolecules must be correctly oriented relative
to each other to bind together when they collide. Thus
the complementary binding sites are aligned correctly
only 0.1% to 5% of the times that the molecules collide.
Many binding reactions between two proteins,
between enzymes and substrates, and between proteins
and larger molecules (eg, DNA) are said to be diffusion
limited in the sense that the rate constant is determined
by diffusion-driven collisions between the reactants.
Thus many association rate constants are in the range of
106 to 107M1s1.
CHAPTER 4 n Biophysical Principles 55
To review, the rate of a second-order reaction is
simply the product of a constant that is characteristic of
In biology, the rates of many bimolecular association
reactions are determined by the rates of diffusion-limited
collisions between the reactants.
Reversible Reactions
Most reactions are reversible, so the net rate of a reaction
is equal to the difference between the forward and reverse
reaction rates. The forward and reverse reactions can be
any combination of first- or second-order reactions. A
reversible conformational change of a protein from A to
A* is an example of a pair of simple first-order reactions:
A  A*
The forward reaction rate is k+ A with units of M s1, and
the reverse reaction rate is k A* with the same units. At
equilibrium, when the net concentrations of A and A* no
longer change,
k+ [ A ] = k [ A*]
and
K eq = k+ k = [ A*] [ A ]
This equilibrium constant Keq is unitless because the
units of concentration and the rate constants cancel
out. Equilibrium constants are designated by upper-
case Ks.
The same reasoning with respect to the equilib-
rium constant applies to a simple bimolecular binding
reaction:
A + B  AB
where A and B are any molecule (eg, enzyme, receptor,
substrate, cofactor, or drug). The forward (binding) reac-
tion is a second-order reaction, whereas the reverse (dis-
sociation) reaction is a first-order reaction. The opposing
reactions are
Rate of association = k+ [ A ][B]
Units: M s 1
Rate of dissociation = k [ AB]
Units: M s 1
The overall rate of the reaction is the forward rate
minus the reverse rate:
Net rate = association rate dissociation rate
= k+ [ A ][B] k [ AB]
Depending on the values of the rate constants and
the concentrations of A, B, and AB, the reaction can go
forward, backward, or nowhere.
56 SECTION II n Chemical and Physical Background
At equilibrium, the forward and reverse rates are (by
definition) the same:
k+ [ A ][B] = k [ AB]
The equilibrium constant for such a bimolecular reaction
can be written in two ways:
Association equilibrium constant:
K a = [ AB] [ A ][B] = k+ k
Units: M 1 = M M M
This is the classical equilibrium constant used in chem-
istry, where the strength of the reaction is proportional
to the numerical value. For bimolecular reactions, the
units of reciprocal molar are difficult to relate to, so
biochemists frequently use the reciprocal relationship:
Dissociation equilibrium constant:
K d = [ A ][B] [ AB] = k k+
Units: M = M M M
When half of the total A is bound to B, the concentra-
tion of free B is simply equal to the dissociation equilib-
rium constant.
Thermodynamic Considerations
The driving force for chemical reactions is the lowering
of the free energy of the system when reactants are
converted into products. The larger the reduction in free
energy, the more completely reactants will be converted
to products at equilibrium. A thorough consideration of
thermodynamics is beyond the scope of this text, but
an overview of this subject is presented to allow the
reader to gain a basic understanding of its power and
simplicity.
The change in Gibbs free energy, G, is simply the
difference in the chemical potential, , of the reactants
(R) and products (P):
G = P R
The chemical potential of a particular chemical species
depends on its intrinsic properties and its concentration,
expressed as the equation
= 0 + RT ln C
where 0 is the chemical potential in the standard state
(1M in biochemistry), R is the gas constant (8.3Jmol1 because the equilibrium constant depends on the ratio
degree1), T is the absolute temperature in degrees
Kelvin, and C is the ratio of the concentration of the
chemical species to the standard concentration. Because
the standard state is defined as 1M, the parameter C has
the same numerical value as the molar concentration,
but is, in fact, unitless. The term RT ln C adjusts for the
concentration. When C = 1, = 0.
Under standard conditions in which 1mol of reactant
is converted to 1mol of product, the standard free
energy change, G0, is
G 0 = 0 P 0 R
However, because most reactions do not take place
under these standard conditions, the chemical potential
must be adjusted for the actual concentrations. This
is done by including the concentration term from the
definition of the chemical potential. An equation for
the free energy change that takes concentrations into
account is
G = 0 P + RT ln[P] 0 R RT ln[R ]
Substituting the definition of G0, we have
G = G 0 + RT ln[P] RT ln[R ] = G 0 + RT ln[P] [R ]
This relationship tells us that the free energy change for
the conversion of reactants to products is simply the free
energy change under standard conditions corrected for
the actual concentrations of reactant and products.
At equilibrium, the concentrations of reactants and
products do not change and the free energy change is
zero, so
0 = G 0 + RT ln[Peq ] [R eq ]
or
G 0 = RT ln[Peq ] [R eq ]
You are already familiar with the fact that the equilib-
rium constant for a reaction is the ratio of the equilib-
rium concentrations of products and reactants. Thus that
relationship can be substituted in this thermodynamic
equation:
G 0 = RT ln K
or
0
K = e G RT
= k + k = [Peq ] [R eq ]
This profound relationship shows how the free energy
change is related to the equilibrium constant. The change
in the standard Gibbs free energy, G0, specifies the ratio
of products and reactants when the reaction reaches
equilibrium, regardless of the rate or path of the reac-
tion. The free energy change provides no information
about whether or not a given reaction will proceed on
a time scale relevant to cellular activities. Nevertheless,
of the rate constants, knowledge of the rate constants
reveals the equilibrium constant and the free energy
change for a reaction. Consider the consequences of
various values of G0:
0
If G0 equals 0, e G RT equals 1, and at equilib-
rium, the concentration of products will equal
the concentration of reactants (or in the case of a

bimolecular reaction, the product of the concentra-
tions of the reactants).
0
If G0 is less than 0, e G RT is greater than 1, and
at equilibrium, the concentration of products will
be greater than the concentration of reactants.
Larger, negative free energy changes will drive the
reaction farther toward products. Favorable reac-
tions have large negative G0 values.
0
If G0 is greater than 0, e G RT is less than 1, and
at equilibrium, the concentrations of reactants will
exceed the concentration of products.
It is sometimes said that a reaction with a positive G0
will not proceed spontaneously. This is not strictly true.
Reactants will still be converted to products, although
relative to the concentration of reactants, the concentra-
tion of products will be small. The size and sign of the
free energy change tell nothing about the rate of a reac-
tion. For example, the oxidation of sucrose by oxygen
is highly favored with a G0 of 5693kJ/mol, but a flash
fire in a sugar bowl is an event rarely, if ever, seen.*
The free energy change is additionally related to two
thermodynamic parameters that are important to the
subsequent discussion of molecular interactions. The
Gibbs-Helmholtz equation is the key relationship:
G = H TS
where H is the change in enthalpy, an approximation
(with a small correction for pressure-volume work) of
the bond energies of the molecules. Thus H is the heat
given off when a bond is made or the heat taken up
when a bond is broken. The change in enthalpy is simply
the difference in enthalpy of reactants and products.
In biochemical reactions, the enthalpy term principally
reflects energies of the strong covalent bonds and of the
weaker hydrogen and electrostatic bonds. If no covalent
bonds change, as in a binding reaction or a conforma-
tional change, H is determined by the difference in
the energy of the weak bonds of the products and
reactants.
The change in entropy, expressed as S, is a measure
of the change in the order of the products and reactants.
The value of the entropy is a function of the number of
microscopic arrangements of the system, including the
solvent molecules. Note the minus sign in front of the
TS term. Reactions are favored if the change in entropy
is positive, that is, if the products are less-well-ordered
than the reactants. Increases in entropy drive reactions
by increasing the negative free energy change. For
example, the hydrophobic effect, which is discussed
later in this chapter, depends on an increase in entropy.
Increases in entropy provide the free energy change for
*Eisenberg D, Crothers D. Physical Chemistry with Applications to
the Life Sciences. Menlo Park, CA: Benjamin Cummings, 1979.
CHAPTER 4 n Biophysical Principles 57
many biologic reactions, especially macromolecular
folding (see Chapters 3 and 12) and assembly (see
Chapter 5).
As emphasized in the case of G, neither the rate of
the reaction nor the path between reactants and prod-
ucts is relevant to the difference in enthalpy or entropy
of reactants and products. The reader may consult a
physical chemistry book for a fuller explanation of these
basic principles of thermodynamics.
Linked Reactions
Many important processes in the cell consist of a single
reaction, but most of cellular biochemistry involves a
series of linked reactions (Fig. 4.3). For example, when
two macromolecules bind together, the complex often
undergoes some type of internal rearrangement or con-
formational change, linking a first-order reaction to a
second-order reaction.
A + B  AB AB  AB*
One of thousands of such examples is GTP binding to a
G protein, causing it to undergo a conformational change
from the inactive to the active state (Figs. 4.6 and 4.7
later).
Similarly, the basic enzyme reaction considered in
most biochemistry books is simply a series of reversible
second- and first-order reactions:
E + S  ES ES  EP EP  E + P
where E is enzyme, S is substrate, and P is product.
These and more complicated reactions can be described
rigorously by a series of rate equations like those
explained previously. For example, enzyme reactions
nearly always involve one or more additional intermedi-
ates between ES and EP, coupled by first-order reactions,
in which the molecules undergo conformational changes.
Linking reactions together is the strategy used by cells
to carry out unfavorable reactions. All that matters is
that the total free energy change for all coupled reactions
is negative. An unfavorable reaction is driven forward
by a favorable reaction upstream or downstream. For
example, a proton gradient across the mitochondrial
A + B A B A* B
Dissociation Favorable conformational
favored change pulls the linked
reaction to the right
FIGURE 4.3 LINKED REACTIONS. Two molecules, A and B, bind
together weakly and then undergo a favorable conformational change.
The binding reaction is unfavorable, owing to the high rate of dissocia-
tion of AB, but the favorable conformational change pulls the overall
reaction far to the right.
58 SECTION II n Chemical and Physical Background
membrane is used as an energy source to drive the
unfavorable reaction producing adenosine triphosphate
(ATP) from adenosine diphosphate (ADP) and inorganic
phosphate (see Fig. 14.5). This proton gradient is derived,
in turn, from the oxidation of chemical bonds of nutri-
ents. To use a macroscopic analogy, a siphon can initially
move a liquid uphill against gravity provided that the
outflow is placed below the inflow, so that the overall
change in energy is favorable.
An appreciation of linked reactions makes it possible
to understand how catalysts, including biochemical
catalystsprotein enzymes and ribozymesinfluence
reactions. They do not alter the free energy change for
reactions, but they enhance the rates of reactions by
speeding up the forward and reverse rates of unfavorable
intermediate reactions along pathways of coupled reac-
tions. Given that the rates of both first- and second-order
reactions depend on the concentrations of the reactants,
the overall reaction is commonly limited by the concen-
tration of the least-favored, highest-energy intermediate,
called a transition state. This might be a strained confor-
mation of substrate in a biochemical pathway. Interac-
tion of this transition state with an enzyme can lower its
free energy, increasing its probability (concentration)
and thus the rate of the limiting reaction. Acceleration
of biochemical reactions by enzymes is impressive.
Enhancement of reaction rates by 10 orders of magni-
tude is common.
Chemical Bonds
Covalent bonds are responsible for the stable architec-
ture of the organic molecules in cells (Fig. 4.4). They are
very strong. CC and CH bonds have energies of
approximately 400kJmol1. Bonds this strong do not
dissociate spontaneously at body temperatures and pres-
sures, nor are the reactive intermediates required to form
these bonds present in finite concentrations in cells. To
overcome this problem, living systems use enzymes,
which stabilize high-energy transition states, to catalyze
formation and dissolution of covalent bonds. Energy for
making strong covalent bonds is obtained indirectly by
coupling to energy-yielding reactions. For example, met-
abolic enzymes convert energy released by breaking
covalent bonds of nutrients, such as carbohydrates,
lipids, and proteins, into ATP (see Fig. 19.4), which
H site partial charges in the oxygen and hydrogen provide the attractive
S > 400 kJ mol1
H
N 300 400 kJ mol1
H C H H oxygens. D, The hydrophobic effect arises when two complementary,
H O 200 300 kJ mol1
N C C apolar surfaces make contact, excluding water molecules that formerly
H OH < 50 kJ mol1
H
FIGURE 4.4 COVALENT BONDS. Bond energies for the amino
acid cysteine.
supplies energy required to form new covalent bonds
during the synthesis of polypeptides. Metabolic path-
ways relating the covalent chemistry of the molecules of
life are covered in depth in many excellent biochem-
istry books.
For cell biologists, four types of relatively weak inter-
actions (Fig. 4.5) are as important as covalent bonds
because they are responsible for folding macromolecules
into their active conformations and for holding mole-
cules together in the structures of the cell. These weak
interactions are (a) hydrogen bonds, (b) electrostatic
interactions, (c) the hydrophobic effect, and (d) van
der Waals interactions. None of these interactions is
particularly strong on its own. Stable bonding between
subunits of many macromolecular structures, between
ligands and receptors, and between substrates and
enzymes is a result of the additive effect of many weak
interactions working in concert.
Hydrogen and Electrostatic Bonds
Hydrogen bonds (Fig. 4.5) occur between a covalently
bound donor H atom with a partial positive charge, +
(the result of electron withdrawal by a covalently bonded
O or N), and an acceptor atom (usually O or N) with a
partial negative charge, . These bonds are highly direc-
tional, with optimal bond energy (12 to 29kJmol1)
when the H atom points directly at the acceptor atom.
Hydrogen bonds are extremely important in the stabiliza-
tion of secondary structures of proteins, such as -helices
and -sheets (see Fig. 3.8), and in the base pairing of
DNA and RNA (see Fig. 3.14).
A. Hydrogen bond D. Hydrophobic effect
C O H N
+
B. Electrostatic bond
C
O H
N
O +H
C. Electrostatic bond
with chelated metal ion
Water excluded
O O from complementary
C C
O Ca2+ O hydrophobic surfaces
FIGURE 4.5 WEAK INTERACTIONS. A, Hydrogen bond. Oppo-
force. B, Electrostatic bond. Atoms with opposite charges are attracted
to each other. C, Ca2+ chelated between two negatively charged
were associated with the surfaces. The increased disorder of the water
increases the entropy and provides the decrease in free energy to drive
the association. van der Waals interactions between closely packed
atoms on complementary surfaces also stabilize interactions.

Electrostatic (or ionic) bonds occur between charged
groups that have either lost or gained a proton (eg,
COO and NH3+). Although these bonds are poten-
tially about as strong as an average hydrogen bond
(20kJmol1), it has been argued that they contribute
little to biological structure. This is because a charged
group is usually neutralized by an inorganic counterion
(such as Na+ or Cl) that is itself surrounded by a cloud
of water molecules. The effect of having the cloud of
water molecules is that the counterion does not occupy
a single position with respect to the charged group on
the macromolecule; consequently, these interactions
lack structural specificity.
Electrostatic interactions come into their own in dis-
sociating biological structures, since like charges on
potential binding surfaces of macromolecules will repel
one another. This allows phosphorylation to control
many biological interactions. Enzymatic introduction of
a negatively charged phosphate group can disrupt an
otherwise stable interaction between two proteins,
whereas removal of phosphate allows the interaction
(see Chapter 25).
Hydrophobic Effect
Self-assembly and other association reactions that involve
the joining together of separate molecules to form more
ordered structures might seem unlikely when examined
from the point of view of thermodynamics. Nonetheless,
many binding reactions are highly favored, and when
such processes are monitored in the laboratory, it can
be shown that S actually increases.
How can association of molecules lead to increased
disorder? The answer is that the entropy of the system
including macromolecules and solventincreases owing
to the loss of order in the water surrounding the macro-
molecules (Fig. 4.5). This increase in the entropy of the
water more than offsets the increased order and
decreased entropy of the associated macromolecules.
Bulk water is a semistructured solvent maintained by
a loose network of hydrogen bonds (see Fig. 3.1).
Water cannot form hydrogen bonds with nonpolar
(hydrophobic) parts of lipids and proteins. Instead,
water molecules form cages or clathrates of exten-
sively H-bonded water molecules near these hydropho-
bic surfaces. These clathrates are more ordered than is
bulk water or water interacting with charged or polar
amino acids.
When proteins fold (see Fig. 12.10), macromolecules
bind together (see Chapter 5), and phospholipids associ-
ate to form bilayers (see Fig. 13.5), hydrophobic groups
are buried in pockets or between interfaces that exclude
water. The highly ordered water formerly associated
with these surfaces disperses into the less-ordered bulk
phase, and the entropy of the system increases.
The increase in the disorder of water that results
when hydrophobic regions of macromolecules are
CHAPTER 4 n Biophysical Principles 59
buried is called the hydrophobic effect. Hydrophobic
interactions are a major driving force, but they would
not confer specificity on an intermolecular interaction
except for the fact that the molecular surfaces must be
complementary to exclude water. The hydrophobic
effect is not a bond per se, but a thermodynamic factor
that favors macromolecular interactions.
van der Waals Interactions
van der Waals interactions occur when adjacent atoms
come close enough that their outer electron clouds just
barely touch. This action induces charge fluctuations
that result in a nonspecific, nondirectional attraction.
These interactions are highly distance dependent,
decreasing in proportion to the sixth power of the sepa-
ration. The energy of each interaction is only about
4kJmol1 (very weak when compared with the average
kinetic energy of a molecule in solution, which is
approximately 2.5kJmol1) and is significant only when
many interactions are combined (as in interactions of
complementary surfaces). Under optimal circumstances,
van der Waals interactions can achieve bonding energies
as high as 40kJmol1.
When two atoms get too close, they strongly repel
each other. Consequently, imperfect fits between inter-
acting molecules are energetically very expensive, pre-
venting association if surface groups interfere sterically
with each other. As a determinant of specificity of mac-
romolecular interactions, this van der Waals repulsion
is even more important than the favorable bonds dis-
cussed earlier, because it precludes many nonspecific
interactions.
Strategy for Understanding
Cellular Functions
One strategy for understanding the mechanism of any
molecular processincluding binding reactions, self-
assembly reactions, and enzyme reactionsis to deter-
mine the existence of the various reactants, intermediates,
and products along the reaction pathway and then to
measure the rate constants for each step. Such an analy-
sis yields additional information about the thermodynam-
ics of each step, as the ratio of the rate constants reveals
the equilibrium constant and the free energy change,
even for transient intermediates that may be difficult or
impossible to analyze separately.
In earlier times, biochemists lacked methods to evalu-
ate the internal reactions along most pathways, but they
could measure the overall rate of reactions, such as the
steady-state rate of conversion of reactants to products
by an enzyme. To analyze these data, they simplified
complex mechanisms using relationships such as the
Michaelis-Menten equation (described in biochemistry
textbooks). Now, abundant supplies of proteins, conve-
nient methods for measuring rapid reaction rates, and
60 SECTION II n Chemical and Physical Background
computer programs that can be used to analyze complex
reaction mechanisms generally make such simplifica-
tions unnecessary.
Analysis of an Enzyme Mechanism:
The Ras GTPase
This section uses a vitally important family of enzymes
called GTPases to illustrate how enzymes work. The
example is Ras, a small GTPase that serves as part of a
biochemical pathway linking growth factor receptors in
the plasma membrane of animal cells to regulation of the
cell cycle. The example shows how to dissect an enzyme
reaction by kinetic analysis and how crystal structures
can reveal conformational changes related to function.
GTPases related to Ras regulate a host of systems, includ-
ing nuclear transport (see Fig. 9.18), protein synthesis
(see Figs. 12.8 and 12.9), vesicular trafficking (see Fig.
21.6), signaling pathways coupled to seven-helix recep-
tors including vision and olfaction (see Fig. 25.9), the
actin cytoskeleton (see Figs. 33.13 and 33.19), and assem-
bly of the mitotic spindle (see Fig. 44.8). This section
gives the reader the background required to understand
the contributions of GTPases to all these processes as
they are presented in the following sections of the book.
Having evolved from a common ancestor, Ras and its
related GTPases share a homologous core domain that
binds a guanine nucleotide and uses a common enzy-
matic cycle of GTP binding, hydrolysis, and product dis-
sociation to switch the protein on and off (Fig. 4.6). The
GTP-binding domain consists of approximately 200 resi-
dues folded into a six-stranded -sheet sandwiched
between five -helices. GTP binds in a shallow groove
formed largely by loops at the ends of elements of sec-
ondary structure. A network of hydrogen bonds between
the protein and guanine base, ribose, triphosphate, and
Mg2+ anchor the nucleotide. Larger GTPases have a core
GTPase domain plus domains required for coupling to
seven-helix receptors (see Fig. 25.9) or regulating protein
synthesis (see Figs. 12.10 and 25.7).
The bound nucleotide determines the conformation
and activity of each GTPase. The GTP-bound conforma-
tion is active, as it interacts with and stimulates effector
proteins. In the example considered here, the Ras-GTP
binds and stimulates a protein kinase, Raf, which relays
signals from growth factor receptors to the nucleus
(see Fig. 27.6). The guanosine diphosphate (GDP)-bound
conformation of Ras is inactive because it does not bind
effectors. Thus, GTP hydrolysis and phosphate dissocia-
tion switch Ras and related GTPases from the active to
the inactive state.
All GTPases use the same enzyme cycle, which
involves four simple steps (Fig. 4.6). GTP binding favors
the active conformation that binds effector proteins.
GTPases remain active until they hydrolyze the bound
A. Ras-GDP B. Ras-GTP
GTP
Switch I
Switch II
GTP
Inactive Active
1
Fast
G GT
GDP
Rate Slow
4 limiting timer 2
GEF GAP
GDI
GD GDP
Fast
3
Pi
FIGURE 4.6 Top, Atomic structures of the small GTPase Ras. GTP
hydrolysis and phosphate dissociation change the conformations of
the switch loops. (For reference, see Protein Data Bank [www.rcsb.org]
files 1Q21 [A] and 121P [B].) Bottom, Generic GTPase cycle. The size
of the arrows indicates the relative rates of the reactions. GAP, GTPase
activating protein; GD, GTPase with bound GDP; GDI, guanine nucleo-
tide dissociation inhibitor; GDP, GTPase with bound GDP and inorganic
phosphate; GEF, guanine nucleotide exchange factor; GT, GTPase with
bound GTP; Pi, phosphate.
GTP. Hydrolysis is intrinsically slow, but binding to effec-
tor proteins or regulatory proteins can accelerate this
inactivation step. GTPases tend to accumulate in the
inactive GDP state, because GDP dissociation is very
slow. Specific proteins catalyze dissociation of GDP,
making it possible for GTP to rebind and activate the
GTPase. Seven-helix receptors activate their associated
G-proteins. Guanine nucleotide exchange proteins
(GEFs) activate small GTPases.
Figure 4.7 illustrates the experimental strategy used
to establish the mechanism of the Ras GTPase cycle.
Step 1: GTP binding. GTP binds rapidly to nucleotide-
free Ras in two linked reactions (Fig. 4.7A). The first
is rapid but reversible association of GTP with Ras.
Second is a slower but highly favorable first-order
conformational change, which produces the fluores-
cence signal in the experiment and accounts for the
 CHAPTER 4 n Biophysical Principles 61

A Ras + mGTP B Ras GTP C Ras GDP

GTP binding GTP hydrolysis GDP release

Ras mGTP Ras GDP P Ras + GDP

Pi release
= active
Ras GDP + Pi
1.0
Extent of reaction 1.0 1.0

+ Cdc24 GEF
plus NF1 GAP
Ras with bound GTP
minus NF1 GAP
Cdc24 GEF
0 0 0
0 0.1 0 0.6 0 2000.0
Time (sec)
FIGURE 4.7 Kinetic dissection of the Ras GTPase cycle using a series of single turnover experiments, in which each enzyme molecule carries
out a reaction only once. A, GTP binding. Nucleotide-free Ras is mixed rapidly with a fluorescent derivative of GTP (mGTP), and fluorescence is
followed on a millisecond time scale. With 100M mGTP (approximately 10% of the cellular concentration), binding is fast (half-time less than
5ms), but the change in fluorescence is slower, approximately 30s1, because it depends on a subsequent, slower conformational change.
Linking the association reaction to this highly favorable (K = 106) first-order conformational change accounts for the exceedingly high affinity
(Kd = ~1011M) of Ras for GTP. Binding and dissociation of GDP are similar. B, GTP hydrolysis and -phosphate dissociation. GTP is mixed with
Ras, and hydrolysis is followed by collecting samples on a millisecond time scale with a quench-flow device, dissociating the products from
the enzyme and measuring the fraction of GTP converted to GDP. The Ras-GDP-P intermediate releases -phosphate spontaneously in a first-
order reaction. A fluorescent phosphate-binding protein is used to measure free phosphate. On this time scale in this figure, Ras alone does not
hydrolyze GTP or dissociated phosphate because the hydrolysis rate constant is 5 105s1, corresponding to a half-time of 1400 seconds. The
GTPase activating protein (GAP) neurofibromin 1 (NF1) at a concentration of 10M increases the rate of hydrolysis to 20s1 and allows observa-
tion of the time course of phosphate dissociation at 8s1. C, GDP dissociation. Ras with bound fluorescent mGDP is mixed with GTP, which
replaces the mGDP as it dissociates. The loss of fluorescence over time gives a rate constant for mGDP dissociation of 0.00002s1. The guanine
nucleotide exchange factor Cdc24Mn at a concentration of 1M increases the rate of mGDP dissociation 500-fold to 0.01s1. (Data from Lenzen
C, Cool RH, Prinz H, etal. Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide
exchange factor Cdc24Mn. Biochemistry. 1998;37:74207430; and Phillips RA, Hunter JL, Eccleston JF, Webb MR. Mechanism of Ras GTPase
activation by neurofibromin. Biochemistry. 2003;42:39563965.)

high affinity of Ras for GTP (Kd typically in the range
of 1011M). The conformation change involves three
segments of the polypeptide chain called switch I,
switch II, and switch III. Folding of these three loops
around the -phosphate of GTP traps the nucleotide
and creates a binding site for the Raf kinase, the down-
stream effector (see Fig. 27.6).
Step 2: GTP hydrolysis. Hydrolysis is essentially irrevers-
ible and slow with a half-time of approximately 4
hours (Fig. 4.7B). Although slow, GTP hydrolysis on
the enzyme is many orders of magnitude faster than
in solution. Like other enzymes, interactions of the
protein with the substrate stabilize the transition
state, a high-energy chemical intermediate between
GTP and GDP. In this transition state, the -phosphate
is partially bonded to both the -phosphate and an
attacking water. Hydrogen bonds between protein
backbone amides and oxygens bridging the - and
-phosphates and on the - and -phosphates stabilize
negative charges that build up on these atoms in the
transition state. Hydrolysis is slow in comparison with
most enzyme reactions, because none of these hydro-
gen bonds is particularly strong. Another hydrogen
bond from a glutamine side chain helps position a
water for nucleophilic attack on the -phosphate. The
importance of this interaction is illustrated by muta-
tions that replace that glutamine with leucine. This
mutation reduces the rate of hydrolysis by orders of
magnitude and predisposes to the development of
many human cancers by prolonging the active state
and thus amplifying growth-promoting signals from
growth factor receptors.
Step 3: Dissociation of inorganic phosphate. After
hydrolysis, the -phosphate dissociates rapidly. This
reverses the conformational change of the three
switch loops, dismantling the binding site for effector
proteins.
Step 4: Dissociation of GDP. On its own, Ras accumu-
lates in the inactive GDP state, because GDP dissoci-
ates extremely slowly with a half-time of 10 hours
(Fig. 4.7C). GTP cannot bind and activate Ras until
GDP dissociates.
Ras and most other small GTPases depend on regula-
tory proteins to stimulate the two slow steps in the
GTPase cycle: GDP dissociation and GTP hydrolysis. For
example, when growth factors stimulate their receptors,
a series of reactions (see Fig. 27.6) brings a guanine
nucleotide exchange factor (GEF) to the plasma
membrane to activate Ras by accelerating dissociation
62 SECTION II n Chemical and Physical Background
of GDP. First the GEF binds Ras-GDP and then favors a
slow conformational change that distorts a part of Ras
that interacts with the -phosphate. This allows GDP to
dissociate on a time scale of seconds to minutes rather
than 10 hours (Fig. 4.7C). Once GDP has dissociated,
nucleotide-free Ras can bind either GDP or GTP. Binding
GTP is more likely in cells, because the cytoplasmic
concentration of GTP (approximately 1mM) is 10 times
that of GDP. GTP binding activates Ras, allowing trans-
mission of the signal to the nucleus.
GTPase-activating proteins (GAPs) turn off Ras
and related GTPases, by binding Ras-GTP and stimulating
GTP hydrolysis, thereby terminating GTPase activation
(Fig. 4.7B). Ras GAPs stabilize the transition state, by
contributing a positively charged arginine side chain that
stabilizes the negative charges on the oxygen bridging
the - and -phosphates and on the -phosphate. GAPs
also help position Gln61 and its attacking water. In the
experiment in the figure, a GAP called neurofibromin
(NF1) binds Ras with a half-time of 3ms (not illustrated)
and stimulates rapid hydrolysis of GTP at 20s1. This is
followed by rate-limiting dissociation of -phosphate
from the Ras-GDP-P intermediate at 8s1 and rapid dis-
sociation of NF1 from Ras at 50s1. NF1 is the product
of a human gene that is inactivated in the disease called
neurofibromatosis. Lacking the NF1 GAP activity to keep
Ras in check, affected individuals develop numerous
neural tumors that disfigure the skin and may compro-
mise the function of the nervous system.
ACKNOWLEDGMENT
We thank Martin Webb for his help with GTPase kinetics
for the second edition.
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