Chapter 8
Uploaded by
A. AsadiChapter 8
Uploaded by
A. AsadiC H A P T E R
Enzymes: Kinetics
Victor W. Rodwell, PhD
8
OBJ E C TI VE S ■ Describe the scope and objectives o enzyme kinetic analysis.
■ Indicate whether ΔG, the overall change in ree energy or a reaction, is
After studying this chapter, dependent on reaction mechanism.
you should be able to: ■ Indicate whether ΔG is a unction o the rates o reactions.
■ Explain the relationship between Keq, concentrations o substrates and products
at equilibrium, and the ratio o the rate constants k1/k–1.
■ Outline how the concentration o hydrogen ions, o enzyme, and o substrate
aect the rate o an enzyme-catalyzed reaction.
■ Utilize collision theory to explain how temperature aects the rate o a
chemical reaction.
■ Dene initial rate conditions and explain the advantage obtained rom
measuring the velocity o an enzyme-catalyzed reaction under these
conditions.
■ Describe the application o linear orms o the Michaelis-Menten equation to
estimate Km and Vmax.
■ Give one reason why a linear orm o the Hill equation is used to evaluate
how substrate-binding infuences the kinetic behavior o certain multimeric
enzymes.
■ Contrast the eects o an increasing concentration o substrate on the kinetics
o simple competitive and noncompetitive inhibition.
■ Describe how substrates add to, and products depart rom, an enzyme that
ollows a ping-pong mechanism.
■ Describe how substrates add to, and products depart rom, an enzyme that
ollows a rapid-equilibrium mechanism.
■ Provide examples o the utility o enzyme kinetics in ascertaining the mode o
action o drugs.
BIOMEDICAL IMPORTANCE and order o the individual steps by which enzymes transorm
substrates into products and, in conjunction with site-directed
A complete and balanced set o enzyme activities is required mutagenesis, kinetic analyses can reveal details o the catalytic
or maintaining homeostasis. Enzyme kinetics, the quantita- mechanism o a given enzyme. In the blood, the appearance or
tive measurement o the rates o enzyme-catalyzed reactions a surge in the levels o particular enzymes serves as clinical indi-
and the systematic study o actors that aect these rates, con- cators or pathologies such as myocardial inarctions, prostate
stitutes a central tool or the analysis, diagnosis, and treatment cancer, and damage to the liver. he involvement o enzymes
o the enzymic imbalances that underlie numerous human in virtually all physiologic processes makes them the targets
diseases. For example, kinetic analysis can reveal the number o choice or drugs that cure or ameliorate human disease.
71
72 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
Applied enzyme kinetics represents the principal tool by transition rom the standard state, one-molar concentrations
which scientists identiy and characterize therapeutic agents o substrates and products, to equilibrium. A more useul bio-
that selectively inhibit the rates o speciic enzyme-catalyzed chemical term is ΔG0′, which deines ΔG0 at a standard state
processes. Enzyme kinetics thus plays a central and critical o 10–7 M protons, pH 7.0. I the ree energy o ormation o
role in drug discovery, in comparative to pharmacodynamics, the products is lower than that o the substrates, the signs o
and in elucidating the mode o action o drugs. ΔG0 and ΔG0′ will be negative, indicating that the reaction as
written is avored in the direction let to right. Such reactions
are reerred to as spontaneous. he sign and the magnitude
CHEMICAL REACTIONS ARE o the ree-energy change determine how ar the reaction will
DESCRIBED USING BALANCED proceed.
EQUATIONS Equation (3) illustrates the relationship between the equi-
librium constant Keq and ΔG0:
A balanced chemical equation lists the initial chemical species
(substrates) present and the new chemical species (products) ΔG0 = –RT ln Keq (3)
ormed or a particular chemical reaction, all in their respective where R is the gas constant (1.98 cal/mol°K or 8.31 J/mol°K)
proportions or stoichiometry. For example, balanced equa- and T is the absolute temperature in degrees Kelvin. Keq is equal
tion (1) indicates that one molecule each o substrates A and B to the product o the concentrations o the reaction products,
reacts to orm one molecule each o products P and Q: each raised to the power o their stoichiometry, divided by the
A+B⇄P+Q (1) product o the substrates, each raised to the power o their
stoichiometry:
he double arrows indicate reversibility, an intrinsic property For the reaction A + B ⇄ P + Q
o all chemical reactions. hus, or reaction (1), i A and B can
orm P and Q, then P and Q can also orm A and B. Desig- [P][Q]
K eq = (4)
nation o a particular reactant as a “substrate” or “product” is [A][B]
thereore somewhat arbitrary since the products or a reaction
written in one direction are the substrates or the reverse reac- and or reaction (5)
tion. he term “products” is, however, oten used to designate
A+A⇄P (5)
the reactants whose ormation is thermodynamically avored.
Reactions or which thermodynamic actors strongly avor [P]
ormation o the products to which the arrow points oten are K eq = (6)
[A]2
represented with a single arrow as i they were “irreversible”:
A+B→P+Q (2) ΔG0 may be calculated rom equation (3) i the molar concen-
trations o substrates and products present at equilibrium are
Unidirectional arrows are also used to describe reactions in known. I ΔG0 is a negative number, Keq will be greater than
living cells where the products o reaction (2) are immediately unity, and the concentration o products at equilibrium will
consumed by a subsequent enzyme-catalyzed reaction or rap- exceed that o the substrates. I ΔG0 is positive, Keq will be less
idly escape the cell, or example, CO2. he rapid removal o than unity, and the ormation o substrates will be avored.
product P or Q thereore eectively precludes occurrence o Note that, since ΔG0 is a unction exclusively o the initial
the reverse reaction, rendering equation (2) functionally irre- and inal states o the reacting species, it can provide inorma-
versible under physiologic conditions. tion only about the direction and equilibrium state o the reac-
tion. ΔG0 is independent o the mechanism o the reaction,
and provides no inormation concerning rates o reactions.
CHANGES IN FREE ENERGY Consequently—and as explained below—although a reaction
DETERMINE THE DIRECTION may have a large negative ΔG0 or ΔG0′, it may nevertheless take
& EQUILIBRIUM STATE OF place at a negligible rate.
CHEMICAL REACTIONS
he Gibbs ree-energy change ΔG (also called either ree THE RATES OF REACTIONS
energy or Gibbs energy) describes in quantitative orm both
the direction in which a chemical reaction will tend to proceed ARE DETERMINED BY THEIR
and the concentrations o reactants and products that will be ACTIVATION ENERGY
present at equilibrium. ΔG or a chemical reaction equals the
sum o the ree energies o ormation o the reaction prod- Reactions Proceed via Transition States
ucts ΔGp minus the sum o the ree energies o ormation o he concept o the transition state is undamental to under-
the substrates ΔGS. A similar but dierent quantity designated standing the chemical and thermodynamic basis o catalysis.
by ΔG0 denotes the change in ree energy that accompanies Equation (7) depicts a group transer reaction in which an
CHAPTER 8 Enzymes: Kinetics 73
entering group E displaces a leaving group L, attached initially For the overall reaction (10), ΔG is the numeric sum o ΔGF
to R: and ΔGD. As or any equation o two terms, it is not possible to
deduce rom their resultant ΔG, either the sign or the magni-
E+R–L⇄E–R+L (7)
tude o ΔGF or ΔGD.
he net result o this process is to transer group R rom L to Many reactions involve several successive transition states,
E. Midway through the displacement, the bond between R and each with an associated change in ree energy. For these reac-
L has weakened but has not yet been completely severed, and tions, the overall ΔG represents the sum o all o the ree-
the new bond between E and R is yet incompletely ormed. energy changes associated with the ormation and decay o all
his transient intermediate—in which neither ree substrate o the transition states. It therefore is not possible to infer
nor product exists—is termed the transition state, E…R…L. from the overall ΔG the number or type of transition states
Dotted lines represent the “partial” bonds that are undergoing through which the reaction proceeds. Stated another way,
ormation and rupture. Figure 8–1 provides a more detailed overall reaction thermodynamics tells us nothing about mecha-
illustration o the transition state intermediate ormed during nism or kinetics.
the transer o a phosphoryl group.
Reaction (7) can be thought o as consisting o two
“partial reactions,” the irst corresponding to the ormation
ΔGF Defines the Activation Energy
(F) and the second to the subsequent decay (D) o the tran- Regardless o the sign or magnitude o ΔG, ΔGF or the over-
sition state intermediate. As or all reactions, characteristic whelming majority o chemical reactions has a positive sign,
changes in ree energy, ΔGF and ΔGD are associated with each which indicates that ormation o the transition state requires
partial reaction: surmounting one or more energy barriers. For this reason, ΔGF
or reaching a transition state is oten termed the activation
E+R–L ⇄E…R…L ΔGF (8) energy, Eact. he ease—and hence the requency—with which
E…R…L ⇄E–R+L ΔGD (9) this barrier is overcome is inversely related to Eact. he thermo-
dynamic parameters that determine how fast a reaction proceeds
E+R–L ⇄E–R+L ΔG = ΔGF + ΔGD (10) thus are the ΔGF values or ormation o the transition state(s)
through which the reaction proceeds. For a simple reaction,
where ∝ means “proportionate to,”
Rate ∝ e–Eact/R (11)
he activation energy or the reaction proceeding in the oppo-
site direction to that drawn is equal to –ΔGD.
NUMEROUS FACTORS AFFECT
REACTION RATE
he kinetic theory—also called the collision theory—o
chemical kinetics states that or two molecules to react, they (1)
must approach within bond-orming distance o one another,
or “collide,” and (2) must possess suicient kinetic energy to
overcome the energy barrier or reaching the transition state.
It thereore ollows that conditions that tend to increase the
FIGURE 8–1 Formation of a transition state intermedi- frequency or energy o collision between substrates will tend
ate during a simple chemical reaction, A + B → P + Q. Shown are to increase the rate o the reaction in which they participate.
three stages o a chemical reaction in which a phosphoryl group is
transerred rom leaving group L (green) to entering group E (blue).
Top: Entering group E (bracket A) approaches the other reactant, Temperature
L-phosphate (bracket B). Notice how the three oxygen atoms linked Raising the ambient temperature increases the kinetic energy
by the triangular lines and the phosphorus atom o the phosphoryl
o molecules. As illustrated in Figure 8–2, the total number
group orm a pyramid. Center: As E approaches L-phosphate, the new
bond between E and the phosphoryl group begins to orm (dotted o molecules whose kinetic energy exceeds the energy barrier
line) as that linking L to the phosphoryl group weakens. These partially Eact (vertical bar) or ormation o products increases rom
ormed bonds are indicated by dotted lines. Bottom: Formation o the low (A) through intermediate (B) to high (C) temperatures.
new product, E-phosphate (bracket P), is now complete as the leaving Increasing the kinetic energy o molecules also increases their
group L (bracket Q) exits. Notice how the geometry o the phosphoryl
rapidity o motion, and thereore the requency with which
group diers between the transition state and the substrate or prod-
uct. The phosphorus and three oxygen atoms that occupy the our they collide. his combination o more requent and more
corners o a pyramid in the substrate and product become coplanar, as highly energetic, and hence productive, collisions increases
emphasized by the triangle, in the transition state. the reaction rate.
74 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
Energy barrier subscripts 1 and –1 reer to the orward and reverse reactions,
respectively:
A B C Rate1 = k1[A]n[B]m (20)
Number of
molecules
Rate–1 = k–1[P] (21)
he sum o the molar ratios o the reactants deines the
kinetic order o the reaction. Consider reaction (5). he stoi-
0
chiometric coeicient or the sole reactant, A, is 2. hereore,
Kinetic energy
the rate o production o P is proportional to the square o [A]
FIGURE 8–2 Representation of the energy barrier for chemical and the reaction is said to be second order with respect to reac-
reactions. (See text or discussion.) tant A. In this instance, the overall reaction is also second order.
hereore, k1 is reerred to as a second-order rate constant.
Reaction (12) describes a simple second-order reaction
Reactant Concentration between two dierent reactants, A and B. he stoichiometric
he requency with which molecules collide is directly pro- coeicient or each reactant is 1. hereore, while the reaction
portionate to their concentrations. For two dierent molecules is second order, it is said to be first order with respect to A and
A and B, the requency with which they collide will double i first order with respect to B.
the concentration o either A or B is doubled. I the concentra- In the laboratory, the kinetic order o a reaction with respect
tions o both A and B are doubled, the probability o collision to a particular reactant, reerred to as the variable reactant or
will increase ourold. substrate, can be determined by maintaining the concentra-
For a chemical reaction proceeding at a constant tempera- tion o other reactants in large excess over the variable reactant.
ture that involves one molecule each o A and B, Under these pseudo-first-order conditions, the concentration o
the “ixed” reactant remains virtually constant. hus, the rate
A+B→P (12) o reaction will depend exclusively on the concentration o the
variable reactant, sometimes also called the limiting reactant.
the raction o the molecules possessing a given kinetic energy he concepts o reaction order and pseudo-irst-order condi-
will be a constant. he number o collisions between mole- tions apply not only to simple chemical reactions but also to
cules whose combined kinetic energy is suicient to produce enzyme-catalyzed reactions.
product P thereore will be directly proportionate to the num-
ber o collisions between A and B, and thus to their molar con- Keq Is a Ratio of Rate Constants
centrations, denoted by the square brackets:
While all chemical reactions are to some extent reversible, at
Rate ∝ [A] [B] (13) equilibrium the overall concentrations o reactants and prod-
ucts remain constant. At equilibrium, the rate o conversion o
Similarly, or the reaction represented by substrates to products thereore equals the rate at which prod-
A + 2B → P (14) ucts are converted to substrates:
which can also be written as Rate1 = Rate–1 (22)
hereore,
A+B+B→P (15)
k1 = [A]n[B]m = k–1[P] (23)
the corresponding rate expression is
and
Rate ∝ [A] [B] [B] (16) k1 [P]
= n m (24)
or k−1 [A] [B]
Rate ∝ [A][B]2 (17) he ratio o k1 to k–1 is equal to the equilibrium constant, Keq.
For the general case, when n molecules o A react with m he ollowing important properties o a system at equilibrium
molecules o B, must be kept in mind:
nA + mB → P (18) 1. he equilibrium constant is a ratio o the reaction rate
constants (not the reaction rates).
the rate expression is
2. At equilibrium, the reaction rates (not the rate constants)
Rate ∝ [A]n [B]m (19) o the orward and back reactions are equal.
Replacing the proportionality sign with an equals sign by 3. he numeric value o the equilibrium constant Keq can be
introducing a rate constant, k, characteristic o the reac- calculated either rom the concentrations o substrates and
tion under study gives equations (20) and (21), in which the products at equilibrium or rom the ratio k1/k–1.
CHAPTER 8 Enzymes: Kinetics 75
4. Equilibrium is a dynamic state. Although there is no net Since the enzyme on both sides o the double arrows is present
change in the concentration o substrates or products, in equal quantity and identical orm, the expression or the
individual substrate and product molecules are continually equilibrium constant,
being interconverted. Interconvertibility can be proved by
adding to a system at equilibrium a trace o radioisotopic [P][Q][Enz]
K eq = (27)
product, which can then be shown to result in the appear- [A][B][Enz]
ance o radiolabelled substrate.
reduces to one identical to that or the reaction in the absence
o the enzyme:
THE KINETICS OF ENZYME [P][Q]
K eq = (28)
[A][B]
CATALYSIS
Enzymes thereore have no eect on Keq.
Enzymes Lower the Activation Energy
Barrier for a Reaction
All enzymes accelerate reaction rates by lowering ΔGF or the or-
MULTIPLE FACTORS AFFECT THE
mation o transition states. However, they may dier in the way RATES OF ENZYME-CATALYZED
this is achieved. While the sequence o chemical steps at the active REACTIONS
site parallels those which occur when the substrates react in the
absence o a catalyst, the environment of the active site low- Temperature
ers ΔGF by stabilizing the transition state intermediates. o put Raising the temperature increases the rate o both uncatalyzed
it another way, the enzyme can be envisioned as binding to the and enzyme-catalyzed reactions by increasing the kinetic energy
transition state intermediate (Figure 8–1) more tightly than it does and the collision requency o the reacting molecules. However,
to either substrates or products. As discussed in Chapter 7, sta- heat energy can also increase the conormational lexing o the
bilization can involve (1) acid–base groups suitably positioned to enzyme to a point that exceeds the energy barrier or disrupting
transer protons to or rom the developing transition state inter- the noncovalent interactions that maintain its three-dimensional
mediate, (2) suitably positioned charged groups or metal ions that structure. he polypeptide chain then begins to unold, or dena-
stabilize developing charges, or (3) the imposition o steric strain ture, with an accompanying loss o the catalytic activity. he
on substrates so that their geometry approaches that o the transi- temperature range over which an enzyme maintains a stable,
tion state. HIV protease (see Figure 7–6) illustrates catalysis by an catalytically competent conormation depends on—and typi-
enzyme that lowers the activation barrier in part by stabilizing a cally moderately exceeds—the normal temperature o the cells
transition state intermediate. in which it resides. Enzymes rom humans generally exhibit sta-
Catalysis by enzymes that proceeds via a unique reaction bility at temperatures up to 45 to 55°C. By contrast, enzymes
mechanism typically occurs when the transition state inter- rom the thermophilic microorganisms that reside in volcanic
mediate orms a covalent bond with the enzyme (covalent hot springs or undersea hydrothermal vents may be stable at
catalysis). he catalytic mechanism o the serine protease chy- temperatures up to or even above 100°C.
motrypsin (see Figure 7–7) illustrates how an enzyme utilizes he temperature coefficient (Q10) is the actor by which the
covalent catalysis to provide a unique reaction pathway pos- rate o a biologic process increases or a 10°C increase in tem-
sessing a more avorable Eact. perature. For the temperatures over which enzymes are stable,
the rates o most biologic processes typically double or a 10°C
rise in temperature (Q10 = 2). Changes in the rates o enzyme-
ENZYMES DO NOT AFFECT Keq catalyzed reactions that accompany a rise or all in body temper-
While enzymes undergo transient modiications during the pro- ature constitute a prominent survival eature or “cold-blooded”
cess o catalysis, they always emerge unchanged at the comple- lie orms such as lizards or ish, whose body temperatures are
tion o the reaction. The presence of an enzyme therefore has dictated by the external environment. However, or mammals
no effect on ΔG0 for the overall reaction, which is a unction and other homeothermic organisms, changes in enzyme reac-
solely o the initial and final states o the reactants. Equation tion rates with temperature assume physiologic importance
(25) shows the relationship between the equilibrium constant or only in circumstances such as ever or hypothermia.
a reaction and the standard ree-energy change or that reaction:
Hydrogen Ion Concentration
ΔG0 = –RT ln Keq (25) he rate o almost all enzyme-catalyzed reactions exhibits a
his principle is perhaps most readily illustrated by includ- signiicant dependence on hydrogen ion concentration. Most
ing the presence o the enzyme (Enz) in the calculation o the intracellular enzymes exhibit optimal activity at pH values
equilibrium constant or an enzyme-catalyzed reaction: between 5 and 9. he relationship o activity to hydrogen
ion concentration (Figure 8–3) relects the balance between
A + B + Enz ⇄ P + Q + Enz (26) enzyme denaturation at high or low pH and eects on the
76 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
100
SH+ E–
v
%
0
Low High FIGURE 8–4 Effect of substrate concentration on the initial
pH velocity of an enzyme-catalyzed reaction.
FIGURE 8–3 Effect of pH on enzyme activity. Consider, or
example, a negatively charged enzyme (E–) that binds a positively (see above), scientists can study the dependence o reaction rate
charged substrate (SH+). Shown is the proportion (%) o SH+ [\\\] and on an individual reactant through the appropriate choice o ixed
o E– [///] as a unction o pH. Only in the cross-hatched area do both and variable substrates. In other words, under pseudo-irst-
the enzyme and the substrate bear an appropriate charge. order conditions the behavior o a multisubstrate enzyme will
imitate one having a single substrate. In this instance, however,
the observed rate constant will be a unction both o the rate
charged state o the enzyme, the substrates, or both. For constant k1 or the reaction and o the concentration o the ixed
enzymes whose mechanism involves acid–base catalysis, the substrate.
residues involved must be in the appropriate state o proton- For a typical enzyme, as substrate concentration is increased,
ation or the reaction to proceed. he binding and recognition vi increases until it reaches a maximum value Vmax (Figure 8–4).
o substrate molecules with dissociable groups also typically When urther increases in substrate concentration ail to increase
involves the ormation o salt bridges with the enzyme. he vi, the enzyme is said to be “saturated” with the substrate. Note
most common charged groups are carboxylate groups (nega- that the shape o the curve that relates activity to substrate con-
tive) and protonated amines (positive). Gain or loss o critical centration (Figure 8–4) is hyperbolic. At any given instant, only
charged groups adversely aects substrate binding and thus substrate molecules that are combined with the enzyme as an
will retard or abolish catalysis. enzyme-substrate (ES) complex can be transormed into product.
Since the equilibrium constant or the ormation o the enzyme-
substrate complex is not ininitely large, only a raction o
ASSAYS OF ENZYME-CATALYZED the enzyme may be present as an ES complex even when the
REACTIONS TYPICALLY MEASURE substrate is present in considerable excess (points A and B o
Figure 8–5). At points A or B, increasing or decreasing [S] there-
THE INITIAL VELOCITY ore will increase or decrease the number o ES complexes with
Most measurements o the rates o enzyme-catalyzed reac- a corresponding change in vi. At point C (Figure 8–5), however,
tions employ relatively short time periods, conditions that essentially all the enzyme is present as the ES complex. Since no
are considered to approximate initial rate conditions. Under ree enzyme remains available or orming ES, urther increases
these conditions, only traces o product accumulate, render- in [S] cannot increase the rate o the reaction. Under these satu-
ing the rate o the reverse reaction negligible. he initial rating conditions, vi depends solely on—and thus is limited
velocity (vi) o the reaction thus is essentially that o the rate by—the rapidity with which product dissociates from the
o the orward reaction. Assays o enzyme activity almost enzyme so that it may combine with more substrate.
always employ a large (103-106) molar excess o substrate over
enzyme. Under these conditions, vi is proportionate to the
concentration o enzyme, that is, it is pseudo-irst-order with THE MICHAELIS-MENTEN & HILL
respect to enzyme. Measuring the initial velocity thereore EQUATIONS MODEL THE EFFECTS
permits one to estimate the quantity o enzyme present in a
biologic sample. OF SUBSTRATE CONCENTRATION
The Michaelis-Menten Equation
SUBSTRATE CONCENTRATION he Michaelis-Menten equation (29) illustrates in mathe-
AFFECTS THE REACTION RATE matical terms the relationship between initial reaction veloc-
ity vi and substrate concentration [S], shown graphically in
In what ollows, enzyme reactions are treated as i they had only Figure 8–4:
a single substrate and a single product. For enzymes with mul-
tiple substrates, the principles discussed below apply with equal Vmax [S]
vi = (29)
validity. Moreover, by employing pseudo-irst-order conditions K m +[S]
CHAPTER 8 Enzymes: Kinetics 77
=S
=E
A B C
FIGURE 8–5 Representation of an enzyme in the presence of a concentration of substrate that is below Km (A), at a concentration
equal to Km (B), and at a concentration well above Km(C). Points A, B, and C correspond to those points in Figure 8–4.
The Michaelis constant Km is the substrate concentration A Linear Form of the Michaelis-Menten
at which vi is half the maximal velocity (Vmax/2) attainable
at a particular concentration of the enzyme. Km thus has
Equation Is Used to Determine Km & Vmax
the dimensions o substrate concentration. he dependence he direct measurement o the numeric value o Vmax, and
o initial reaction velocity on [S] and Km may be illustrated thereore the calculation o Km, oten requires impracti-
by evaluating the Michaelis-Menten equation under three cally high concentrations o substrate to achieve saturating
conditions. conditions. A linear orm o the Michaelis-Menten equa-
tion circumvents this diiculty and permits Vmax and Km to
1. When [S] is much less than Km (point A in Figures 8–4 and be extrapolated rom initial velocity data obtained at less
8–5), the term Km + [S] is essentially equal to Km. Replac- than saturating concentrations o the substrate. Start with
ing Km + [S] with Km reduces equation (29) to equation (29),
Vmax [S]
Vmax [S] V [S] V vi = (29)
vi = vi ≈ max ≈ max [S] (30) K m +[S]
K m +[S] Km Km
invert
where ≈ means “approximately equal to.” Since Vmax and Km 1 K m +[S]
are both constants, their ratio is a constant. In other words, = (33)
vi Vmax [S]
when [S] is considerably below Km, vi is proportionate to k[S].
he initial reaction velocity thereore is directly proportional actor
to [S].
1 Km [S]
2. When [S] is much greater than Km (point C in Figures 8–4 = + (34)
vi Vmax [S] Vmax[S]
and 8–5), the term Km + [S] is essentially equal to [S].
Replacing Km + [S] with [S] reduces equation (29) to and simpliy
Vmax [S] V [S] 1 Km 1 1
vi = vi ≈ max ≈ Vmax (31) = + (35)
K m +[S] [S] vi Vmax [S] Vmax
hus, when [S] greatly exceeds Km, the reaction velocity is Equation (35) is the equation or a straight line, y = ax + b,
maximal (Vmax) and unaected by urther increases in the sub- where y = 1/vi and x = 1/[S]. A plot o 1/vi as y as a unction
strate concentration. o 1/[S] as x thereore gives a straight line whose y intercept
3. When [S] = Km (point B in Figures 8–4 and 8–5): is 1/Vmax and whose slope is Km/Vmax. Such a plot is called a
double reciprocal or Lineweaver-Burk plot (Figure 8–6).
Vmax [S] Vmax[S] Vmax Setting the y term o equation (36) equal to zero and solving
vi = = = (32) or x reveals that the x intercept is –1/Km:
K m +[S] 2[S] 2
− b −1
Equation (32) states that when [S] equals Km, the initial veloc- 0 = ax + b; therefore, x = = (36)
a Km
ity is hal-maximal. Equation (32) also reveals that Km is—and
may be determined experimentally rom—the substrate con- Km can be calculated rom the slope and y intercept, but is per-
centration at which the initial velocity is hal-maximal. haps most readily calculated rom the negative x intercept.
78 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
1 Slope =
Km For certain enzymes, once substrate binds to the active
vi Vmax site, it is converted to product and released so rapidly as to
render these events eectively instantaneous. For these excep-
tionally eicient catalysts, the rate-limiting step in catalysis is
– K1 1 the ormation o the ES complex. Such enzymes are said to
m
Vmax be diffusion-limited, or catalytically perect, since the astest
0
possible rate o catalysis is determined by the rate at which
1
[S] molecules move or diuse through the solution. Examples o
enzymes or which kcat/Km approaches the diusion limit o
FIGURE 8–6 Double-reciprocal or Lineweaver-Burk plot of 108-109 M–1s–1 include triosephosphate isomerase, carbonic
1/vi versus 1/[S] used to evaluate Km and Vmax. anhydrase, acetylcholinesterase, and adenosine deaminase.
In living cells, the assembly o enzymes that catalyze suc-
he greatest virtue o the Lineweaver-Burk plot resides in cessive reactions into multimeric complexes can circumvent
the acility with which it can be used to determine the kinetic the limitations imposed by diusion. he geometric relation-
mechanism o an enzyme inhibitor (see below). However, in ships o the enzymes in these complexes are such that the
using a double-reciprocal plot to determine kinetic constants, it substrates and products do not diuse into the bulk solution
is important to avoid the introduction o bias through the cluster- until the last step in the sequence o catalytic steps is complete.
ing o data at low values o 1/[S]. his bias can be readily avoided Fatty acid synthetase extends this concept one step urther by
in the laboratory as ollows. Prepare a solution o substrate whose covalently attaching the growing substrate atty acid chain to
dilution into an assay will produce the maximum desired con- a biotin tether that rotates rom active site to active site within
centration o the substrate. Now prepare dilutions o the stock the complex until synthesis o a palmitic acid molecule is com-
solution by actors o 1:2, 1:3, 1:4, 1:5, etc. Data generated using plete (see Chapter 23).
equal volumes o these dilutions will then all on the 1/[S] axis at
equally spaced intervals o 1, 2, 3, 4, 5, etc. A single-reciprocal plot Km May Approximate a Binding Constant
such as the Eadie-Hostee (vi vs vi/[S]) or Hanes-Wool ([S]/vi vs he ainity o an enzyme or its substrate is the inverse o
[S]) plot can also be used to minimize data clustering. the dissociation constant Kd or dissociation o the enzyme-
substrate complex ES:
The Catalytic Constant, kcat
E + S←
→ ES
k1
(38)
k−1
Several parameters may be used to compare the relative activ-
ity o dierent enzymes or o dierent preparations o the k−1
same enzyme. he activity o impure enzyme preparations Kd = (39)
k1
typically is expressed as a specific activity (Vmax divided by the
protein concentration). For a homogeneous enzyme, one may Stated another way, the smaller the tendency o the enzyme
calculate its turnover number (Vmax divided by the moles o and its substrate to dissociate, the greater the ainity o the
enzyme present). However, i the number o active sites pres- enzyme or its substrate. While the Michaelis constant Km
ent is known, the catalytic activity o a homogeneous enzyme oten approximates the dissociation constant Kd, this should
is best expressed as its catalytic constant, kcat (Vmax divided by not be assumed, or it is by no means always the case. For a
the number o active sites, St): typical enzyme-catalyzed reaction:
Vmax k1
kcat = (37)
E + S← → ES k
2
→E + P (40)
St k−1
Since the units o concentration cancel out, the units o kcat are he value o [S] that gives vi = Vmax/2 is
reciprocal time.
k−1 + k2
[S] = = Km (41)
Catalytic Efficiency, kcat/Km k1
By what measure should the eiciency o dierent enzymes, When k–1 >> k2, then
dierent substrates or a given enzyme, and the eiciency
with which an enzyme catalyzes a reaction in the orward and k–1 + k2 ≈ k–1 (42)
reverse directions be quantiied and compared? While the and
maximum capacity o a given enzyme to convert substrate to k1
product is important, the beneits o a high kcat can only be [S] ≈ = Kd (43)
k−1
realized i Km is suiciently low. hus, catalytic efficiency o
enzymes is best expressed in terms o the ratio o these two Hence, 1/Km only approximates 1/Kd under conditions where
kinetic constants, kcat/Km. the association and dissociation o the ES complex are rapid
CHAPTER 8 Enzymes: Kinetics 79
relative to catalysis. For the many enzyme-catalyzed reactions 1
or which k–1 + k2 is not approximately equal to k–1, 1/Km will
vi
underestimate 1/Kd.
Vmax –
vi
0 Slope = n
The Hill Equation Describes the
Log
Behavior of Enzymes That Exhibit –1
Cooperative Binding of Substrate
While most enzymes display the simple saturation kinetics
–4 S50 –3
depicted in Figure 8–4 and are adequately described by the
Michaelis-Menten expression, some enzymes bind their sub- Log [S]
strates in a cooperative ashion analogous to the binding o
FIGURE 8–8 A graphical representation of a linear form of
oxygen by hemoglobin (see Chapter 6). Cooperative behavior the Hill equation is used to evaluate S50, the substrate concen-
is an exclusive property o multimeric enzymes that bind sub- tration that produces half-maximal velocity, and the degree of
strate at multiple sites. cooperativity n.
For enzymes that display positive cooperativity in bind-
ing the substrate, the shape o the curve that relates changes
in vi to changes in [S] is sigmoidal (Figure 8–7). Neither the site then enhances the ainity o the remaining sites to bind
Michaelis-Menten expression nor its derived plots can be additional substrate. he greater the value or n, the higher
used to evaluate cooperative kinetics. Enzymologists there- the degree o cooperativity and the more markedly sigmoidal
ore employ a graphic representation o the Hill equation will be the plot o vi versus [S]. A perpendicular dropped rom
originally derived to describe the cooperative binding o O2 the point where the y term log vi/(Vmax – vi) is zero intersects
by hemoglobin. Equation (44) represents the Hill equation the x-axis at a substrate concentration termed S50, the substrate
arranged in a orm that predicts a straight line, where k′ is a concentration that results in hal-maximal velocity, S50, thus is
complex constant: analogous to the P50 or oxygen binding to hemoglobin (see
Chapter 6).
log vi
= n log[S]− log k′ (44)
Vmax − vi
KINETIC ANALYSIS
Equation (44) states that when [S] is low relative to k′, the ini- DISTINGUISHES COMPETITIVE
tial reaction velocity increases as the nth power o [S].
A graph o log vi/(Vmax – vi) versus log[S] gives a straight line FROM NONCOMPETITIVE
(Figure 8–8). he slope o the line, n, is the Hill coefficient, an INHIBITION
empirical parameter whose value is a unction o the number, Inhibitors o the catalytic activities o enzymes provide both
kind, and strength o the interactions o the multiple substrate- pharmacologic agents and research tools or the study o the
binding sites on the enzyme. When n = 1, all binding sites mechanism o enzyme action. he strength o the interac-
behave independently and simple Michaelis-Menten kinetic tion between an inhibitor and an enzyme depends on orces
behavior is observed. I n is greater than 1, the enzyme is said important in protein structure and ligand binding (hydrogen
to exhibit positive cooperativity. Binding o substrate to one bonds, electrostatic interactions, hydrophobic interactions,
and van der Waals orces; see Chapter 5). Inhibitors can be
classiied on the basis o their site o action on the enzyme, on
whether they chemically modiy the enzyme, or on the kinetic
parameters they inluence. Compounds that mimic the tran-
sition state o an enzyme-catalyzed reaction (transition state
analogs) or that take advantage o the catalytic machinery o
vi
an enzyme (mechanism-based inhibitors) can be particularly
potent inhibitors. Kinetically, we distinguish two classes o
inhibitors based on whether raising the substrate concentra-
tion does or does not overcome the inhibition.
Competitive Inhibitors Typically
0 [S]
Resemble Substrates
he eects o competitive inhibitors can be overcome by rais-
FIGURE 8–7 Representation of sigmoid substrate saturation ing the concentration o substrate. Most requently, in compet-
kinetics. itive inhibition, the inhibitor (I) binds to the substrate-binding
80 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
H C COO– –2H H C COO–
– –
OOC C H OOC C H
Succinate
H dehydrogenase
Succinate Fumarate
FIGURE 8–9 The succinate dehydrogenase reaction.
portion o the active site thereby blocking access by the sub-
strate. he structures o most classic competitive inhibitors
thereore tend to resemble the structure o a substrate, and
FIGURE 8–10 Lineweaver-Burk plot of simple competitive
thus are termed substrate analogs. Inhibition o the enzyme inhibition. Note the complete relie o inhibition at high [S]
succinate dehydrogenase by malonate illustrates competitive (ie, low 1/[S]).
inhibition by a substrate analog. Succinate dehydrogenase cat-
alyzes the removal o one hydrogen atom rom each o the two
methylene carbons o succinate (Figure 8–9). Both succinate 1/Vmax, this pattern indicates that when 1/[S] approaches 0,
and its structural analog malonate (–OOC—CH2—COO–) can vi is independent of the presence of inhibitor. Note, how-
bind to the active site o succinate dehydrogenase, orming ever, that the intercept on the x-axis does vary with inhibitor
an ES or an EI complex, respectively. However, since malo- concentration and that, since –1/K′m is smaller than –1/Km,
nate contains only one methylene carbon, it cannot undergo K′m (the “apparent Km”) becomes larger in the presence o
dehydrogenation. increasing concentrations o the inhibitor. hus, a competi-
he ormation and dissociation o the EI complex is a tive inhibitor has no effect on Vmax but raises K′m, the appar-
dynamic process described by ent Km for the substrate. For a simple competitive inhibition,
the intercept on the x-axis is
k1
E − I← →E + I (45)
k−1
−1 [I]
x= 1 + (47)
or which the equilibrium constant Ki is K m Ki
[E][I] k1 Once Km has been determined in the absence o inhibitor, Ki
Ki = = (46)
[E − I] k−1 can be calculated rom equation (47). Ki values are used to
compare dierent inhibitors o the same enzyme. he lower
In eect, a competitive inhibitor acts by decreasing the num-
the value or Ki, the more eective the inhibitor. For example,
ber of free enzyme molecules available to bind substrate,
the statin drugs that act as competitive inhibitors o 3-hydroxy-
that is, to form ES, and thus eventually to form product, as
3-methylglutaryl coenzyme A (HMG-CoA) reductase (see
described below.
Chapter 26) have Ki values several orders o magnitude lower
A competitive inhibitor and substrate exert reciprocal
than the Km or the substrate, HMG-CoA.
eects on the concentration o the EI and ES complexes. Since
the ormation o ES complexes removes ree enzyme avail-
able to combine with the inhibitor, increasing [S] decreases the
concentration o the EI complex and raises the reaction veloc-
Simple Noncompetitive Inhibitors
ity. he extent to which [S] must be increased to completely Lower Vmax but Do Not Affect Km
overcome the inhibition depends on the concentration o the In strict noncompetitive inhibition, binding o the inhibitor
inhibitor present, its ainity or the enzyme (Ki), and the ain- does not aect binding o the substrate. Formation o both EI
ity, Km, o the enzyme or its substrate. and enzyme inhibitor substrate (EIS) complexes is thereore
possible. However, while the enzyme-inhibitor complex can
still bind the substrate, its eiciency at transorming substrate
Double-Reciprocal Plots Facilitate the to product, relected by Vmax, is decreased. Noncompetitive
Evaluation of Inhibitors inhibitors bind enzymes at sites distinct rom the substrate-
Double-reciprocal plots typically are used both to distinguish binding site and generally bear little or no structural resem-
between competitive and noncompetitive inhibitors and to blance to the substrate.
simpliy evaluation o inhibition constants. vi is determined For simple noncompetitive inhibition, E and EI possess
at several substrate concentrations both in the presence and in identical ainity or the substrate, and the EIS complex gen-
the absence o the inhibitor. For classic competitive inhibition, erates product at a negligible rate (Figure 8–11). More com-
the lines that connect the experimental data point converge plex noncompetitive inhibition occurs when binding o the
at the y-axis (Figure 8–10). Since the y intercept is equal to inhibitor does aect the apparent ainity o the enzyme or
CHAPTER 8 Enzymes: Kinetics 81
1
vi
[S]
v
–K i [I]
1
FIGURE 8–11 Lineweaver-Burk plot for simple noncompeti- vi
tive inhibition.
[S]
the substrate, causing the lines to intercept in either the third
or ourth quadrants o a double-reciprocal plot (not shown).
While certain inhibitors exhibit characteristics o a mixture o
competitive and noncompetitive inhibition, the evaluation o –K i [I]
these inhibitors exceeds the scope o this chapter.
FIGURE 8–12 Applications of Dixon plots. Top: Competi-
tive inhibition, estimation o Ki. Bottom: Noncompetitive inhibition,
Dixon Plot estimation o Ki.
A Dixon plot is sometimes employed as an alternative to the
Lineweaver-Burk plot or determining inhibition constants.
he initial velocity (vi) is measured at several concentrations tightly bound inhibitors requires specialized kinetic equations
o the inhibitor, but at a ixed concentration o the substrate that incorporate the concentration o enzyme to estimate Ki
(S). For a simple competitive or noncompetitive inhibitor, a or IC50 and to distinguish competitive rom noncompetitive
plot o 1/vi versus inhibitor concentration [I] yields a straight tightly bound inhibitors.
line. he experiment is repeated at dierent ixed concentra-
tions o the substrate. he resulting set o lines intersects to
the let o the y-axis. For competitive inhibition, a perpendicu-
Irreversible Inhibitors “Poison” Enzymes
lar dropped to the x-axis rom the point o intersection o the In the above examples, the inhibitors orm a dissociable,
lines gives –Ki (Figure 8–12, top). For noncompetitive inhibi- dynamic complex with the enzyme. Fully active enzyme can
tion, the intercept on the x-axis is –Ki (Figure 8–12, bottom). thereore be recovered simply by removing the inhibitor rom
Pharmaceutical publications requently employ Dixon plots to the surrounding medium. However, a variety o other inhibi-
illustrate the comparative potency o competitive inhibitors. tors act irreversibly by chemically modiying the enzyme.
hese modiications generally involve making or breaking
covalent bonds with aminoacyl residues essential or substrate
IC50 binding, catalysis, or maintenance o the enzyme’s unctional
A less rigorous alternative to Ki as a measure o inhibitory conormation. Since these covalent changes are relatively
potency is the concentration o inhibitor that produces 50% stable, an enzyme that has been “poisoned” by an irreversible
inhibition, IC50. Unlike the equilibrium dissociation constant inhibitor such as a heavy metal atom or an acylating reagent
Ki, the numeric value o IC50 varies as a unction o the speciic remains inhibited even ater the removal o the remaining
circumstances o substrate concentration, etc under which it inhibitor rom the surrounding medium.
is determined.
Mechanism-Based Inhibition
Tightly Bound Inhibitors “Mechanism-based” or “suicide” inhibitors are specialized
Some inhibitors bind to enzymes with such high ainity, Ki ≤ substrate analogs that contain a chemical group that can be
10–9 M, that the concentration o inhibitor required to measure transormed by the catalytic machinery o the target enzyme.
Ki alls below the concentration o enzyme typically present in Ater binding to the active site, catalysis by the enzyme gen-
an assay. Under these circumstances, a signiicant raction o erates a highly reactive group that orms a covalent bond to
the total inhibitor may be present as an EI complex. I so, this and blocks the function of a catalytically essential residue.
violates the assumption, implicit in classical steady-state kinet- he speciicity and persistence o suicide inhibitors, which are
ics, that the concentration o ree inhibitor is independent o both enzyme-speciic and unreactive outside the conines o
the concentration o enzyme. he kinetic analysis o these the enzyme’s active site, render them promising leads or the
82 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
development o enzyme-speciic drugs. he kinetic analysis o two substrates add in a random or in a compulsory order. For
suicide inhibitors lies beyond the scope o this chapter. Nei- random-order reactions, either substrate A or substrate B may
ther the Lineweaver-Burk nor the Dixon approach is appli- combine irst with the enzyme to orm an EA or an EB com-
cable since suicide inhibitors violate a key boundary condition plex (Figure 8–13, center). For compulsory-order reactions,
common to both approaches, namely that the activity o the A must irst combine with E beore B can combine with the
enzyme does not decrease during the course o the assay. EA complex. One explanation or why some enzymes ollow
a compulsory-order mechanism can be ound in Koshland’s
induced it hypothesis: the addition o A induces a conorma-
MOST ENZYME-CATALYZED tional change in the enzyme that aligns residues that recognize
REACTIONS INVOLVE TWO OR and bind B.
MORE SUBSTRATES
While several enzymes have a single substrate, many others Ping-Pong Reactions
have two—and sometimes more—substrates and products. he term “ping-pong” applies to mechanisms in which one
he undamental principles discussed above, while illustrated or more products are released rom the enzyme beore all the
or single-substrate enzymes, apply also to multisubstrate substrates have been added. Ping-pong reactions involve cova-
enzymes. he mathematical expressions used to evaluate mul- lent catalysis and a transient, modiied orm o the enzyme
tisubstrate reactions are, however, complex. While a detailed (see Figure 7–4). Ping-pong Bi-Bi reactions are oten reerred
analysis o the ull range o multisubstrate reactions exceeds to as double displacement reactions. he group undergoing
the scope o this chapter, some common types o kinetic transer is irst displaced rom substrate A by the enzyme to
behavior or two-substrate, two-product reactions (termed orm product P and a modiied orm o the enzyme (F). he
“Bi-Bi” reactions) are considered below. subsequent group transer rom F to the second substrate B,
orming product Q and regenerating E, constitutes the second
Sequential or Single-Displacement displacement (Figure 8–13, bottom).
Reactions
In sequential reactions, both substrates must combine with Most Bi-Bi Reactions Conform to
the enzyme to orm a ternary complex beore catalysis can Michaelis-Menten Kinetics
proceed (Figure 8–13, top). Sequential reactions are some- Most Bi-Bi reactions conorm to a somewhat more complex
times reerred to as single-displacement reactions because the orm o Michaelis-Menten kinetics in which Vmax reers to
group undergoing transer is usually passed directly, in a sin- the reaction rate attained when both substrates are present
gle step, rom one substrate to the other. Sequential Bi-Bi reac- at saturating levels. Each substrate has its own characteristic
tions can be urther distinguished on the basis o whether the Km value, which corresponds to the concentration that yields
hal-maximal velocity when the second substrate is present
A B P Q at saturating levels. As or single-substrate reactions, double-
reciprocal plots can be used to determine Vmax and Km. vi is
E EA EAB-EPQ EQ E measured as a unction o the concentration o one substrate
(the variable substrate) while the concentration o the other
A B P Q substrate (the ixed substrate) is maintained constant. I the
lines obtained or several ixed-substrate concentrations are
EA EQ plotted on the same graph, it is possible to distinguish a ping-
pong mechanism, which yields parallel lines (Figure 8–14),
E EAB-EPQ E
rom a sequential mechanism, which yields a pattern o inter-
EB EP secting lines (not shown).
B A Q P
Product inhibition studies are used to complement
kinetic analyses and to distinguish between ordered and ran-
A P B Q dom Bi-Bi reactions. For example, in a random-order Bi-Bi
reaction, each product will act as a competitive inhibitor in
E EA-FP F FB-EQ E
the absence o its coproducts regardless o which substrate is
designated the variable substrate. However, or a sequential
FIGURE 8–13 Representations of three classes of Bi-Bi reac- mechanism (Figure 8–13, top), only product Q will give the
tion mechanisms. Horizontal lines represent the enzyme. Arrows pattern indicative o competitive inhibition when A is the vari-
indicate the addition o substrates and departure o products. Top: able substrate, while only product P will produce this pattern
an ordered Bi-Bi reaction, characteristic o many NAD(P)H-dependent
oxidoreductases. Center: a random Bi-Bi reaction, characteristic o with B as the variable substrate. he other combinations o
many kinases and some dehydrogenases. Bottom: a ping-pong reac- product inhibitor and variable substrate will produce orms o
tion, characteristic o aminotranserases and serine proteases. complex noncompetitive inhibition.
CHAPTER 8 Enzymes: Kinetics 83
Increasing irst and oremost, to select appropriate assay conditions or
[S2] detecting the presence o an inhibitor. he concentration o
substrate, or example, must be adjusted such that suicient
product is generated to permit acile detection o the enzyme’s
activity without being so high that it masks the presence o
an inhibitor. Second, enzyme kinetics provides the means or
1 quantiying and comparing the potency o dierent inhibitors
vi and deining their mode o action. Noncompetitive inhibitors
are particularly desirable, because—by contrast to competitive
inhibitors—their eects can never be completely overcome by
increases in substrate concentration.
Most Drugs Are Metabolized In Vivo
1
[S1] Drug development oten involves more than the kinetic
evaluation o the interaction o inhibitors with the target
FIGURE 8–14 Lineweaver-Burk plot for a two-substrate enzyme. In order to minimize its eective dosage, and hence
ping-pong reaction. Increasing the concentration o one substrate the potential or deleterious side eects, a drug needs to be
(S1) while maintaining that o the other substrate (S2) constant alters resistant to degradation by enzymes present in the patient
both the x and y intercepts, but not the slope.
or pathogen, a process termed drug metabolism. For exam-
ple, penicillin and other β-lactam antibiotics block cell wall
synthesis in bacteria by irreversibly inactivating the enzyme
KNOWLEDGE OF ENZYME alanyl alanine carboxypeptidase-transpeptidase. Many bac-
KINETICS, MECHANISM, teria, however, produce β-lactamases that hydrolyze the crit-
AND INHIBITION AIDS DRUG ical β-lactam unction in penicillin and related drugs. One
strategy or overcoming the resulting antibiotic resistance is
DEVELOPMENT to simultaneously administer a β-lactamase inhibitor with a
Many Drugs Act as Enzyme Inhibitors β-lactam antibiotic.
Metabolic transormation is sometimes required to convert
he goal o pharmacology is to identiy agents that can: an inactive drug precursor, or prodrug, into its biologically
1. Destroy or impair the growth, invasiveness, or develop- active orm (see Chapter 47). 2′-Deoxy-5-luorouridylic acid,
ment o invading pathogens. a potent inhibitor o thymidylate synthase, a common target
o cancer chemotherapy, is produced rom 5-luorouracil via
2. Stimulate endogenous deense mechanisms. a series o enzymatic transormations catalyzed by a phospho-
3. Halt or impede aberrant molecular processes triggered by ribosyl transerase and the enzymes o the deoxyribonucleo-
genetic, environmental, or biologic stimuli with minimal side salvage pathway (see Chapter 33). he eective design and
perturbation o the host’s normal cellular unctions. administration o prodrugs requires knowledge o the kinetics
and mechanisms o the enzymes responsible or transorming
By virtue o their diverse physiologic roles and high degree them into their biologically active orms.
o substrate selectivity, enzymes constitute natural targets or
the development o pharmacologic agents that are both potent
and speciic. Statin drugs, or example, lower cholesterol SUMMARY
production by inhibiting the enzyme HMG-CoA reductase ■ Te study o enzyme kinetics—the actors that aect the rates
(see Chapter 26), while emtricitabine and tenoovir diso- o enzyme-catalyzed reactions—reveals the individual steps by
proxil umarate block replication o the human immunodei- which enzymes transorm substrates into products.
ciency virus by inhibiting the viral reverse transcriptase (see ■ ΔG, the overall change in ree energy or a reaction, is
Chapter 34). Pharmacologic treatment o hypertension oten independent o reaction mechanism and provides no
includes the administration o an inhibitor o angiotensin- inormation concerning rates o reactions.
converting enzyme, thus lowering the level o angiotensin II, a ■ Keq, a ratio o reaction rate constants, may be calculated rom
vasoconstrictor (see Chapter 41). the concentrations o substrates and products at equilibrium or
rom the ratio k1/k–1. Enzymes do not aect Keq.
Enzyme Kinetics Defines Appropriate ■ Reactions proceed via transition states, or whose ormation
Screening Conditions the activation energy is reerred to as ΔGF. emperature,
hydrogen ion concentration, enzyme concentration, substrate
Enzyme kinetics plays a crucial role in drug discovery. Knowledge concentration, and inhibitors all aect the rates o enzyme-
o the kinetic behavior o the enzyme o interest is necessary, catalyzed reactions.
84 SECTION II Enzymes: Kinetics, Mechanism, Regulation, & Role o Transition Metals
■ Measurement o the rate o an enzyme-catalyzed reaction ■ In ping-pong reactions, one or more products are released
generally employs initial rate conditions, or which the virtual rom the enzyme beore all the substrates have been added.
absence o product eectively precludes the reverse reaction ■ Applied enzyme kinetics acilitate the identifcation,
rom taking place. characterization, and elucidation o the mode o action o
■ Linear orms o the Michaelis-Menten equation simpliy drugs that selectively inhibit specifc enzymes.
determination o Km and Vmax. ■ Enzyme kinetics plays a central role in the analysis and
■ A linear orm o the Hill equation is used to evaluate the optimization o drug metabolism, a key determinant o drug
cooperative substrate-binding kinetics exhibited by some ecacy.
multimeric enzymes. Te slope n, the Hill coecient, reects
the number, nature, and strength o the interactions o the
substrate-binding sites. A value o n greater than 1 indicates REFERENCES
positive cooperativity. Cook PF, Cleland WW: Enzyme Kinetics and Mechanism. Garland
■ Te eects o simple competitive inhibitors, which typically Science, 2007.
resemble substrates, are overcome by raising the concentration Copeland RA: Evaluation of Enzyme Inhibitors in Drug Discovery.
o the substrate. Simple noncompetitive inhibitors lower Vmax John Wiley & Sons, 2005.
but do not aect Km. Cornish-Bowden A: Fundamentals of Enzyme Kinetics. Portland
■ For simple competitive and noncompetitive inhibitors, the Press Ltd, 2004.
inhibitory constant Ki is equal to the dissociation constant or Dixon M: Te graphical determination o Km and Ki. Biochem J
the relevant enzyme-inhibitor complex. A simpler and less 1972;129:197.
rigorous term widely used in pharmaceutical publications Fersht A: Structure and Mechanism in Protein Science: A Guide to
or evaluating the eectiveness o an inhibitor is IC50, the Enzyme Catalysis and Protein Folding. Freeman, 1999.
concentration o inhibitor that produces 50% inhibition under Schramm, VL: Enzymatic transition-state theory and transition-
the particular circumstances o an experiment. state analogue design. J Biol Chem 2007;282:28297.
Segel IH: Enzyme Kinetics. Wiley Interscience, 1975.
■ Substrates may add in a random order (either substrate may Wlodawer A: Rational approach to AIDS drug design through
combine frst with the enzyme) or in a compulsory order structural biology. Annu Rev Med 2002;53:595.
(substrate A must bind beore substrate B).