etic Tests for Determining from the change in z

H O ! CO y
slopeH CO
at any given
2
[I]. Knowing [I]
fluid) in glaucoma. Carbonic anhydrase plays an important
2 2 3

ibition Mechanisms and !,

Thiswe can process
is an important calculate KIof from
in the transport the role
CO from the expression
in these and other secretory processes, because it par-
ticipates in regulating the pH and bicarbonate content of 2
tissues to the lungs.
228If 10.0Chapter
!g of 6pure carbonic anhydrase
Enzymes
catalyzes the hydration of 0.30 g of CO2 in 1 min at 37 "C at [I] several body fluids. The experimental curve of initial reaction
double-reciprocal plot (see Box 61) offers an ! ! 1 " ## 8885d_c06_205 velocity (as percentage of V ) versus [S] for the carbonic
2/2/04 2:51 PM Page 205 mac76 mac76:385_reb:
Vmax, what is the turnover number (kcat) of carbonic anhy- max

210 Chapter 6 Enzymes K respondanhydrase reaction is illustrated below (upper curve). When
drase (in units of min#1)? I
y way of determining whether an enzyme inhibitor Other heterotropic allosteric enzymes
tivator by an increase in V with little change
thetoexperiment
an ac- iswith
in K curve iserotropic
an increase
repeated in K (Fig.
in the presence
allosteric
629b, lower curve). Het-
of acetazolamide,
enzymesoftherefore
the curvesshow different
0.5

For uncompetitive
13. Deriving a Rate Equation
and
for Competitive
mixed Inhibi-
inhibition,
the lower
similar
obtained. From an inspection max 0.5

ompetitive, uncompetitive, or mixed. Two sets of tion The rate equation (Fig. 629c). A negative
for an enzyme subjectmodulator
to competi- (an inhibitor)
and your may kinds
knowledge of of responses
the in their
kinetic properties of substrate-activity
competitive curves,

plots of rate data give the

tive inhibition is produce a more sigmoid substrate-saturation
families of lines
curve,enzyme
and mixed
shown
because some
inhibitors,
in
have inhibitory
determine the naturemodulators,
of the in- some have 6.3 Enzyme Kinetics as an Approach to Understanding Mechanism 205

experiments are carried out, with the enzyme V [S]

hibition by acetazolamide.
activatingExplain your reasoning.
modulators, and some have both.
max

Figures BOX 62 2 and(a)WORKING 3.

V $ %%
K ""[S] Changes
! [SV] "IN
K when in623)axis
V BIOCHEMISTRY holds forintercepts
all en-100 Some signal
0
1

centration held constant in each set. In the first

(Eqn Regulatory Enzymes Undergo Reversible
Vmax [S] V0 " Vmax m m 0 2 max
zymes V that follow Michaelis-Menten kinetics. (The most V0 " max

changes in Vmaximportant and K .

Km
Beginning with a new definition of total enzyme
exceptions as
to Michaelis-Menten kinetics are Covalent Modification
m discussed in Section 6.5.) How-
[S] is also held constant, permitting measurement the regulatory
[E] $ [E] ! [EI] ! [ES]
enzymes, No inhibitor
In another important class of regulatory enzymes, ac-

V (% of Vmax)
t
ever, the Michaelis-Menten equation does not depend
Kinetic Tests for Determining and the definitions of "onand

V0 (!M/min)
from the change in slope attivity anyisgiven [I].byKnowing [I]

V0 (&M/min)
modulated covalent modification of the en-
he effect of increasing inhibitor concentration [I] the K
relatively simple two-step
text, reaction
derive mechanism

( )
provided in the I
proposed by Michaelis and Menten (Eqn 610). Many 50 zyme molecule. Modifying groups include phosphoryl,
Inhibition Mechanisms the rate equation above.
1Use the derivation
enzymes K
that follow
and
m
of the we can
!,1Michaelis-
VMichaelis-Menten
calculate
%
kinetics have
KI from
adenylyl,
the 1 expression
2 Vmax
uridylyl, methyl, and adenosine diphosphate 1

the initial rate V0 (not shown). In the second set,

The double-reciprocal plot (see 14.
Menten equation as a guide.
Box 61) offers V0an!
quite different
Vwith six or[S]
reaction mechanisms, "andVenzymes that
steps of- ! ! 1 " ##
[I] groups
ribosyl
Acetazolamide
(Fig. 630). These groups are generally
2 max

Irreversible catalyze
Inhibition of an max
reactions Enzyme eight
Many en- max
identifiable
linkedKI to and removed from the regulatory enzyme by
s held constant but [S] is varied. The results are
easy way of determining whether an enzyme inhibitor zymes are inhibited ten exhibit the same steady-state kinetic behavior. Even
irreversibly by heavy metal ions such
though Equation 623 holds true for many enzymes,
as separate enzymes.
Hg , Cu , or Ag , which can react with essential For uncompetitive
sulfhydryl and 0.2mixed An example inhibition,
of0.6
an enzyme similar1 by methylation
0.8 regulated
2! 2! !

ted as 1/V0 versus 1/[S]. is competitive, uncompetitive, or groups mixed. to formTwo sets
mercaptides: of
both the magnitude and the real meaning
K one enzyme to the next. This is an
plots
of V and
the[I]isfamilies
0.4 Km
max

(mof ) rate data 2to give [S]of

(m lines shown in
K can differ from the methyl-accepting chemotaxis protein of bacteria. 0.5

rate experiments are carried out, with EnzSH the enzyme

8885d_c06_190-237 1/27/04 7:13 AM
important limitation of the %!
Page 209 mac76 mac76:385_reb:
[S] steady-state approach en- ) [S] (mM)
This protein is part of a system that permits a bacterium
m
! M
M

Figure 1 shows a set of double-reciprocal plots, ! Ag On EnzSAg ! H !

zyme kinetics. The Figures
parameters V 2 and andK can3. Changes toin axis intercepts signal
be ob-
FIGURE 612 Dependence of initial velocity on substrate concen-
swim toward an attractant (such as a sugar) in solu- max m
concentration held constant in each set. In the
(b) first
tained experimentally for any given enzyme, but by
tration. This graph shows the kinetic parameters that define the limits! !
8885d_c06_190-237 1/27/04 The affinity of Ag for sulfhydryl groups is so great that Ag
changes in mLVmax 17. The Effectstion
and the Km.
of Reversible
and away Inhibitors
from repellent Derive the ex- The methylat-
chemicals.
obtained in the absence of inhibitor and twoheld atconstant, permitting 7:13 AM Pagethemselves 210 V mac76 mac76:385_reb:
%!1.5

)
to titrate OSH groupsthey provide little information about
of theKinetics
6.3 Enzyme curve at Approach
as an high and low [S]. Mechanism
to Understanding Km '' [S] and the [S]
At low [S], 209 max
set, [S] is also can be used measurement quantitatively. To 10.0 pression for the effect of a reversible inhibitor on observed

number, rates, or chemical nature of discrete steps in
of a solution containingthe
V0 $ M/min
ing agent is S-adenosylmethionine (adoMet) (see Fig.
term in the denominator of the Michaelis-Menten equation (Eqn 69)
1.0reaction.
mg/mL Steady-state
of a pure enzyme, an in-
kinetics nevertheless Kis the
ing the rate of individual reaction steps reveals howbecomes
en- (apparent K $ "K /"&). Start with Equation 630 and
(a) Competitive inhibition

erent concentrations of a competitive of inhibitor.

the effect of In-
m m m
increasing inhibitor concentration [I] 1818b).
insignificant. ADP-ribosylation is an especially
The equation simplifies to V0 " Vmax[S]/Km and interesting re-

( )
ergy is used by a specific enzyme, which is an impor-
vestigator added just enough AgNO to completely inactivate compare theandstatement that apparent K is equivalent to the [S] at

1 V (! /min)1
E#S ES E#P
tant component of the overall reaction mechanism. V In0aexhibits a linear dependence on [S], as observed here. At high [S],
standard language by which biochemists
number of cases investigators have been able to record 1 %!1 #
K 1
action, observed
% in only a few proteins; the ADP-ribose 3
m
m

on the initial rate V0 (not shown).theInenzyme. the Asecond set, ofcatalytic

total of characterize
0.342 !molthe AgNO efficiencies
was required. which
where [S] '' V $ V /2"&.
Km, the Km term in the denominator of the Michaelis-
S S
of enzymes. I

V ! V " Vfrom nicotinamide adenine dinucleotide (NAD) " 3 0 max

is[S]
the rates of every individual step in a multistep enzy-

asing [I] results in a family of lines with

[I] is a common matic reaction. Some examples of the applicationMenten of equation derived
becomes insignificant and the equation simplifies to 0 max max

M
K

held constant but [S] is varied. The results are V and K Figure 6 12V shows a simple
presteady state kinetics are included in the descrip- I

Interpreting (see Fig. 841). This type of modification occurs for the
V0 " Vmax;EIthis is consistent with the plateau observed at high [S]. The

(
tions of specific enzymes in Section 6.4. max m 1

0
I I max
2

rcept on the 1/V0 axis but with different

plotted as slopes.
graphical method for obtaining an approximate value #
bacterial enzyme dinitrogenase reductase, resulting in
Michaelis-Menten equation is therefore consistent with the observed
1/V0 versus 1/[S].
Enzymes Are Subject to Reversible
[I] process of biological nitrogen
(b) Uncompetitive
of V0 on [S],inhibition
for K . A more convenient procedure, using a double- dependence
regulation
and the shape of the curve is defined by the
% !of2the important
or Irreversible Inhibition m
reciprocal plot, is presented in Box 61. The K can
Enzyme inhibitors are molecular agents that interfereterms VmaxE/K #S
m at low [S] and Vmax at high [S].
ES E#P

Figure
ause the intercept on the 1/V0 axis equals 1/V210 1 shows a set of double-reciprocal plots,
max,
m
with catalysis, slowing or halting enzymatic reactions. fixation. Diphtheria toxin and cholera toxin are enzymes
#

Chapter 6 Enzymes vary greatly from enzyme to enzyme, and even for dif-
Enzymes catalyze virtually all cellular processes, so it
should not be surprising that enzyme inhibitors are
I
S
that catalyze the ADP-ribosylation (and inactivation) of
ferent substrates of the same enzyme (Table 6 6). The
one obtained in the absence of inhibitor and two
On dividing by V , we obtain
atsometimes %!1.5

)
KI$ S
among the most important pharmaceutical agents max

know that Vmax is unchanged by thedifferent

presence of a

V0 $ M/min
term is K K used (often Kinappropriately) as an
known. For example, aspirin (acetylsalicylate) inhibits key cellular enzymes or proteins. Diphtheria toxin acts
ESI
"
0.5 0.5
#
0.5
the enzyme that catalyzes the first step in the synthe- 1 [S]
concentrations of a competitive1inhibitor. In- of the affinity [S]
indicator of an
(m enzyme
) for its substrate.
sis of prostaglandins, compounds involved in many on and inhibits elongation factor 2, a protein involved in
!! " !!
I
(622) M

1
processes, including some that produce pain. The study %!1 2 Km # [S]
The actual meaning of K (b) depends on specific aspects
mpetitive inhibitor. That is, regardless of the con-
I

'with protein biosynthesis. Cholera toxin acts on a G protein m

creasing [I] results inK a, wefamily
get K # of[S] "lines Km a common
of enzyme inhibitors also has provided valuable infor- S
Solving for 2[S], or
(c) of the reaction mechanism such as the number and rel-
mation about enzyme mechanisms and has helped de- m m
that is part of a signaling pathway (see Fig. 1239), lead-

(
fine some metabolic pathways. There are two broad
ative rates of the individual steps. For reactions with (c) Mixed inhibition

tration of a competitive inhibitor, intercept on the 1/V0 axis but with different slopes. V
classes of enzyme inhibitors: reversible and irreversible.
1
a sufficiently ing to several physiological responses including a massive max

BOX 62 WORKING IN BIOCHEMISTRY Km " [S],E #when V0 " !!Vmax (623)

1
E#S ES P
two steps,
Reversible Inhibition One common type of reversible # # 2
loss of body fluids and, sometimes, death.
Because the intercept on the 1/Vdefinition
0 axisof equals
K : K is 1/Vmax,
inhibition is called competitive (Fig. 615a). A com- I I

This is a very useful, practical petitive inhibitor competes with the substrate for the S k2 # k$1

h substrate concentration will always we displace Vthe

Phosphorylation is the most common type of regu- m m Km " !! (624)

V0 (!M/min)
active site of an enzyme. While the inhibitor (I) occu- S
KI KI$
k1
know that equivalent to the substrate concentration at which V is
max Vis .unchanged by the presence of kais rate-limiting, k %% k and
pies the active site it prevents binding of the substrate

( )
0
When to the enzyme. Many competitive inhibitors are com-
K reduces to latory modification; one-third to one-half of all proteins
EI # S ESI

1
one-half "
1
2 2 m

1
pounds that resemble the substrate and combine with max I I $1

bitor from the enzymes active site. Aboveinhibitor.

competitive the
KineticThat Testsis,for Determining
regardless
The Michaelis-Menten equation (Eqn 69) can be
of theKcon-
V
k /k , which is defined as the dissociation
the enzyme to form an EI complex, but without leading

, of the ES complex. Where these K

' from the change
constant,
to catalysis. Even fleeting combinations of this type will
in a eukaryotic cell are phosphorylated. Some proteins
in slope at any given [I]. Knowing [I] S $1 1
max

a measure of the [S]

conditions hold, K have only one phosphorylated residue, others have sev- m
algebraically transformed into versions that are useful
mtheM
reduce the efficiency of the enzyme. By taking into ac- S d m

centration of inathecompetitive
practical determinationinhibitor, a sufficiently and !, we can eral,calculate fromofthe
KIdozens sites expression
I I

Inhibition Mechanisms
count the molecular geometry of inhibitors that resem-

ph is the rearrangement of Equation 628 on which of K and V (Box 61) does represent
V affinity of
ble the substrate, we can reach conclusions about which enzyme and a few have for phosphorylation.
m max max
FIGURE 615 Three types of reversible inhibition. (a) Competitive
and, as we describe later, in the analysis of parts of the normal substrate bind to the enzyme. Com-
This mode of covalent modification is central to a large
high substrateinhibitor
concentration will always displace
TABLE 66 the [I]
inhibitors bind to the enzymes active site. (b) Uncompetitive inhibitors
0 PM Page 25 mac76 mac76:385_reb: petitive inhibition can be analyzed quantitatively by #
action (see Box 62 on page 210). bind at a separate site, but bind only to the ES complex. KI is the equi-

plot is based. The value of ! can inhibitor

be calculated The double-reciprocal plot (see Box 61)
K for Some Enzymes
offers an
and Substrates
steady-state kinetics. In the presence of a competitive

( )
FIGURE 1
active2 site.
1
V number of regulatory! ! " pathways,
# # and we therefore dis-
librium constant for inhibitor binding to E; KI$ is the equilibrium con- m
Uncompetitive
Above the inhibition.
inhibitor, the Michaelis-Menten equation (Eqn 69)

fromKinetic
theParameters
enzymes becomes 1 1 KI
stant for inhibitor binding to ES. (c) Mixed inhibitors bind at a sepa- 2 max

cuss it in considerable detail.

rate site, but may bind to either E or ES.

Are Used to Compare Enzyme Substrate

K easy way of determining whether an enzyme inhibitor
Vmax [S]
V0 ! ""
K (m )
(628)
M
[S] mM 0.5
m

graph is the rearrangement adding more substrate. of

628 Equation
onorwhich 0.4 For uncompetitive and mixed inhibition, similar
!Km # [S]
Enzyme Activities Hexokinase (brain) ATP [S] (mM)
where
is competitive, uncompetitive,
ply by When [S]
mixed. Two
far exceeds [I],
sets of Phosphoryl Groups Affect the Structure and

( )
D-Glucose ( )
the probability that an inhibitor molecule will bind to 0.05
the plot25is based. plots ofinhibition.
rate Catalytic
data give the families of lines shown in
rateThe value of ! specific
cancarried
be calculated
[I] [E][I]
! ! 1 # "" It is
andimportant
KI ! "" to distinguish between the
the enzyme is minimized and the reaction exhibits a
FIGURE 2 forUncompetitive
1 ! & Km 1 " 1 experiments
equation and theare out, with the enzyme
[EI]
Activity of Proteins
KI
1.3 Physical Foundations FIGURE 629 D-Fructose
Substrate-activity
normal Vmax. However, the [S] at which V0 ! "12" Vmax, the curves 1.5 allosteric
representative
Michaelis-Menten

( )
Figures enzymes2 and 3. Changes in axis intercepts signal
Equation 628 describes the important features of apparent Km, increases in the presence of inhibitor by $
Carbonic anhydrase
enzymes. Three examples HCO 26
of complex responses of allosteric
Ksigmoid
competitive inhibition. The experimentally determined the factor !. This effect on apparent Km, combined with 3
kinetic mechanism on which it was originally
V0 Vmax [S] V concentration
The equation describesheld
variable !Km, the Km observed in the presence of the
constant in each
Chymotrypsin 1 set.(a)&In the 1
first
Glycyltyrosinylglycine
the absence of an effect on Vmax, is diagnostic of com-
m & %
108 enzyme, in The attachment of phosphoryl groups to specific amino

( )
based. the kinetic be- to their modulators. The curve of a homotropic
(a) Mechanical examplemax changes in Vacid and K .
inhibitor, is often called the apparent Km. petitive inhibition and is readily revealed in a double-

1of ! &isKmany
Because the inhibitor binds reversibly to the enzyme,
set, a[S] also " 1constant,
1 held V0 Note ! measurement
N-Benzoyltyrosinamide
-Lactose [S]
VDmax
reciprocal plot (Box 62). The equilibrium constant for
permitting "(stimulatory)
Vmax
2.5 maxresidues ofma protein is catalyzed by protein kinases;

( )
havior great m enzymes,
the competition can be biased to favor the substrate sim- and all en- which the substrate also serves as a positive
inhibitor binding, KI, can be obtained from the same plot. modulator,
4.0
1 curve &ofKm removal
8885d_c01_027 12/20/03 7:08 AM Page zymes 27 mac76 !-Galactosidase
V0that Vmax
exhibit amac76:385_reb:
[S] dependence
hyperbolic Vmax or activator. the resemblance to the oxygen-saturation 1 of& %phosphoryl groups is catalyzed by protein
Threonine dehydratase L-Threonine
G > 0 G < 0
of
V onthe effect of Page
increasing27 mac76inhibitor hemoglobin concentration [I]of a positive5.0modulator
( )
8885d_c01_027 of12/20/03 [S] are said toAM
7:08 follow 0 Michaelis- mac76:385_reb: (see Fig. 512). (b) The effects V0 ! (+) [S] " Vmax KThe addition of a phosphoryl group to
Vmax phosphatases.
e in the affairs of men, Work Loss of
Menten kinetics. The practical rule that
on the initial rate V0 (not shown). In the
and a negative second
modulator (#) on anset,
allosteric enzyme in which K a Ser, 1Thr, ! or V Tyrmresidue1 introduces
"V
% a bulky, charged
at the flood, leads on to fortune; done potential
energy of is altered without a change in V . The central curve shows the groupVinto 0 a region
maxthat[S]
was only moderately polar. The
max
0.5

raising
[I] is held constant but [S] is substrate-activity varied. The results are
max
he voyage of their life object position
relationship without a modulator. (c) A less common oxygen atoms of a phosphoryl group can hydrogen-bond
hallows and in miseries.
hat this passage says overtly, it has [I] plotted as 1/V0 versus 1/[S]. [I]type of modulation, in which V is altered and K is nearly constant. with one or several groups in a protein, commonly the
%!2 [I]
max 0.5

anings. It not only reflects8885d_c01_027 12/20/03 7:08 & !AM3 Page 27 mac76 Figure 1 shows a &set
mac76:385_reb: ! 3of double-reciprocal plots, [I] [I]

)
a complex
)

V0 $ M/min
V0 $ M/min

nts in the play, it also echoes the plays

)

one obtained in the absence of inhibitor and two at 1.3 Physical Foundations %!1.5 27
)

)
Endergonic Exergonic
V0 $ M/min

, ambition, and the demands of lead-

V0 $ M/min
1
1.3 Physical Foundations 27
V0 $ M/min

ed with Shakespeares understanding

different concentrations &! of2a competitive inhibitor. In-
1
1

1
%!1
, it is very rich in information. (b) Chemical example
& ! 2
(
1

the 125 letters making up this quota- Reaction 2: creasing [I] results in a family of lines with a common
(

1
vor the reaction. This increases &by!orders of magnitude

(
ed to fall into a completely random, ATP ADP " Pi
1
(

Reaction 3:
Stored Other
the intercept onproductive
the 1/V0collisions axis butbetween with different slopes. nutrients
1

Glucose " ATP

as shown in the following box, they
probability of reac-

1
glucose 6-phosphate " ADP
cellular work
vor the reaction. This increases by orders theofof magnitude
(

meaning whatsoever.
& !1 Because intercept on andthe several
1/V0 axis equalsStored 1/Vmax,
Free energy, G

Reaction 1: tants. As a result these factors others, Other

1

o the
Glucose " Pi
probability
glucose 6-phosphate of productive discussed collisionsin between
Chapter 6, reac-
Noenzyme-catalyzed
inhibitor
we know that Vmax is unchanged by12the presence of a1.3 foods reactions nutrients Ingested cellular No inhibitor
work Complex
t

a
Physical Foundations 27
f
t G G
2 3
1 biomolecules
tants. As a result of thesecommonly factors and
proceed 1several at rates others,
greater & Km thanregardless
10 times of the con-
s
h

l e s
competitive inhibitor. SlopeThat is,
e a d
I

'K
r
t !
than theVuncatalyzed Ingested ComplexMechanical
d

m
No inhibitor faster reactions. Vmax
W

G
discussed in Chapter 6, enzyme-catalyzed reactions No inhibitor Solar
1
a
max
o f s
e
f i n i
c
G = G G 3 1"
centration
2
Cellular catalysts of aare,competitive inhibitor, a sufficiently
12with a few exceptions, pro- foods photons biomoleculeswork
m ad
i

( )
commonly proceed at rates greater than 10RNA 1 times
& Kincreases
( )
PMo Page 211 mac76 mac76:385_reb: high(In substrate
some cases,1concentration molecules will havealways catalyticdisplace the
1 the 1 1
m a
:52
i e

teins.
d
n
r

vor reaction. This m by orders of magnitude

( )
a gh
Slopethe
o l Reaction coordinate Osmotic
i

faster than !uncatalyzedroles, reactions. [S] mMenzymes Other[S]work mM 1 1

o

T
Stored
o

inhibitor from the active

Againsite. with Above Solar the Mechanical
n
l
Vthe 126 Energy coupling in mechanical V
as discussed in Chapters 26 and 27.)
t
n a
n

s e l a FIGURE max probability of productive collisions between reac- [S] mM

i

and chemical
nutrients
o

max photons cellularworkwork

y

t
Cellular catalysts are, with a few exceptions, pro-
f
a few graphexceptions,
is the each
rearrangement enzyme catalyzes
of Equationa specific628 on which
h
s

e s processes. (a) The downward motion of an object releases potential

e

oi
l
i
FIGURE
energy made 1 avail-Competitive inhibition. FIGURE 3
r
Mixed inhibition.
e

tants. As amotion, result ofcases,these factors andand several others,

if

h e
a

( )
energy that can do mechanical work. The potential
teins.
downward(In some reaction,
(pink), RNA the molecules eachhave reaction in a cell is catalyzed by a
catalytic
w

( )
d

v n
plot is based. The value of ! can be calculated FIGURE 2 Osmotic
s

Uncompetitive inhibition.
d

can be coupled to the1 of different enzymes are Ingested 1

t O
e
able by spontaneous an exergonic process
r u t
1inupward 1 Complex
f
u different enzyme. Thousands
discussed Chapter in6,Chaptersenzyme-catalyzed reactions
o

ject (blue). (b) Inroles, as discussed 26 required

and 27.) Again with
endergonic movement of another ob-

[S] foods [S] mM work

biomolecules
mproceed
reaction 1, the formation of glucose 6-phosphate
M (P ) yields a product of higher therefore by each
12 cell. The multiplicity of en-

( )
125 letters contain little or no infor-
are very rich in entropy. Such con- 6.3
commonly
from glucose
a fewFor exceptions,
and inorganic phosphate
than theEnzyme
two reactants. Kinetics as reaction,
ati
an Approach
rates
each to greater
enzyme
zymes,
Understandingtheir than
catalyzes 10
specificity
Mechanism
times
a 1(the
specific K211
&ability 1to"discriminate
1
( )
energy this endergonic !G is pos- m
itive. Infaster
reaction 2, thethanexergonic the
! & Km 1
led to the conclusion that information
reaction, and breakdown uncatalyzed
of
each reactions
adenosine
reaction
triphos- reactions.
in
between a cell is catalyzed
reactants), andV their by a [S]at the
Vsusceptibility Vto regula- the Solareyes are particularly ADP
Mechanical 1 &%
phate (ATP) can drive an endergonic reaction when the two A medical therapy based on competition ac- sensitive
V0 ! Vto formaldehyde.
0 max max
RE
fact, the 1branch Competitive
of mathematics calledinhibition.
gy; information has been called neg-
are coupled. The Cellular
different catalysts
enzyme. are,
Thousands with tionaof few
give theFIGURE
exceptions,
cells
different capacity
enzymes
3lower
pro-
to Mixed
are
inhibition.
activation barriers photons " work max [S]
"V
max
ry, which is basic to the programming
exergonic reaction has
tive siteselectively.
a large, negative free-energy
change (!G ), and the endergonic reaction has a smaller, positive free-
is used toThis treatselectivity
patientsiswho have ingested
crucial for the effective
Ethanol competes CataboliceffectivelyHPO4 2# withAnabolicmethanol as an alter-
zyme converts it to acetaldehyde. teins.
The ther- (In some cases, RNA molecules have catalytic
2

therefore required
methanol, by each
a solvent cell. The multiplicity of en- reaction reaction
offound in gas-line antifreeze. Thespecific
liver re- native substrate for alcohol dehydrogenase. The effect of
rs, is closely related to thermodynamic energy change (!G ). The third reaction accomplishes the sum of re-
ganisms are highly ordered, nonran-
1

TABLE 69arithmetic Effectsregulation of cellular

Reversible processes.
Inhibitors on By allowing pathways Osmotic pathways
anol poisoning is slow intravenous infusion
actions 1 and 2, and the free-energy change,
roles, !G .as
and zymes, discussed
!Gtheir
!G , is the
the in
specificity Chapters 3

(the 26 and
ability 27.) to Again discriminate with methanol work
mmensely rich in information and thus sum of !G 1 Because
2 enzyme
is negative, overall
and proceeds spontaneously.Apparent Vmax and Apparent Km
3 alcohol
reaction actionsdehydrogenase
to proceed at converts
significant rates at particular to ethanol is much
[I] (exergonic)like that ATP of a (endergonic)
competitive inhibitor, with
a rate that maintains a controlled is exergonic concen-
a few exceptions, eachand enzyme catalyzes a to specific ADP
between reactants),
formaldehyde, their
times,
which susceptibility
enzymes
is damaging determine regula-
how matter
many tissues. and Blind-
energy& are ! 3 the distinction that ethanol is also a substrate for alcohol [I]
)

bloodstream for several hours. This slows

V0 $ M/min

channeled cell Vinto cellularApparent

activities.
)

reaction, tion give andcells each

Inhibitor
ness thereaction
type
iscapacity
a common in toa lower
Apparentresult is ofcatalyzed
activation
methanol by
barriers Kam
ingestion, because dehydrogenase"and its concentration will decrease over
V0 $ M/min

A
of medical
nreaction formaldehyde, therapy
lessening based
the danger Figureon 126bcompetition at the ac- max
the eyes are particularly sensitive 2#to formaldehyde.
1

releases free energy, which is reactions. (a type of graph called a reac- The thousands of enzyme-catalyzed chemical reac-
& ! 2 Catabolic HPO Anabolic
different diagram)enzyme.
selectively. This Thousands
selectivity of
Vis different
crucial forenzymes the Km effective are 4
1

neys
do work. filter out thearemethanol
Such reactions exergonic; totionbecoordinate
excreted illustrates Nonethis principle for the tions
max in cells are functionally organized into many se- reaction reaction
tive site is used
to products isto treat
conversion patients who have ingested Ethanol competes effectively with methanol as an alter-
(

ee energy from reactants of glucose to glucose 6-phosphate, the first

the urine. step intherefore regulation required of
Competitive by The
of cellular each cell.
processes. VmaxThe
quences Byofmultiplicity
allowing
consecutive of en-
specific
!Kreactions, re-called pathways, in pathways pathways
(

egative value. Endergonic reactions re- the pathway for oxidation glucose. sim- m
& !1
anol, typesaofsolvent
fr energy, reversible inhibition, found in gas-line
zymes,
uncompet- actions their antifreeze.
to proceed
specificity The
at liver
significant
(the which ability rates
the productto native
ofatone
discriminate substrate
particular
reaction becomes the for alcohol
reactant (exergonic) dehydrogenase. The
(endergonic) effect of
1

and their values are posi- plest way to produce glucose 6-phosphate would be:
!G
nical processes, only part of the energy
Uncompetitive V max /!$ K m/!$ ATP
ADP
ed, chemical
thoughreactions oftencandefined be used to in terms of one- theirin the/!$ next. Some pathways degrade organic nutrients
Reaction 1: Glucose " P On glucose 6-phosphate
gonic between times, enzymes
reactants), Mixed determine
i
and how
Vmax matter
susceptibility and energy
to!K m/!$ are
regula-
me.ymes,
In livingalcohol
are in practice observed
systems some energydehydrogenase
is dissi- only with
channeled
converts (endergonic; !G

into
methanol
is positive)

cellular
1

HPO . activities.
into to
simple end ethanol
products in is
order much
to extract No like
chemical that
inhibitor of a competitive inhibitor, withNo inhibitor
lost to increasing entropy. (P is an tion giveforcells
abbreviation inorganic the capacity
phosphate, toenergy
lowerandactivation
2#
convert it into barriers
a form useful 1 to the cell; to- & Km "
ldehyde, which is damaging
i

to many tissues.
4
ing
anisms, two ormechanical
as in the more substrates.
example in An beuncom-
Dont concerned about the structure of these com-
Thethem thousands later of
Blind-
enzyme-catalyzed the
chemical distinction
reac- that Slope ethanol
! Catabolic is also a
HPO substrate
2#
4
CO2
Anabolic for alcohol
exergonic reaction can be coupled to
ibitor (Fig. 615b) binds at apounds siteselectively.
now; we describe
distinct This selectivity
in detail in the isgether
crucial thesefor the
degradative, effective
free-energy-yielding
Vmax reactions Vmax reaction NH
reaction
3
s a common
eaction to drive otherwise unfavorable
strate active site and, result
unlike aof methanol tions inofcells ingestion,
book.) This reaction does not occur spontaneously; !G
regulation
competitive cellular are processes.
functionally because are By organized
designated
allowing dehydrogenase
into
catabolism.
specific many re-se-
Other pathwaysand start withits pathways
concentrationSimwill pathways decrease s over

( )
H 2O

( )
met when [S] ! !K m/!$.
smallThus, precursorapparent moleculesKm ! and !K /!$. 1them
mconvert 1 to pro- or 1 1
ds only to the ES complex. In the presence actions quences to proceed of consecutive at reactions,
significant called
rates at pathways,
particular in (exergonic) ple (endergonic) urs
This expression is simpler gressivelywhenlarger eitherand ! or more!$ iscomplex [S] mM includ-
1.0 (formolecules, ATP p r o d u cts, p r e c [S] mM
mpetitive inhibitor, the Michaelis-Menten which
times, enzymes the product
determine
uncompetitive of one reaction
how
or competitive matter
ing proteins becomesand
inhibitors),
and the
energy
nucleic reactant
as acids. are
summarizedSuch synthetic pathways,
tered to channeled in the next. in Table
into Some
cellular 69.pathways activities.degrade FIGURE 1 organic Competitive nutrients
inhibition. FIGURE 128 The FIGURE central role 3 of Mixed
ATP in inhibition.
metabolism. ATP is the
shared chemical intermediate linking energy-releasing to energy-
Vmax [S] into simple
The thousands endpractice,
In ofproducts
enzyme-catalyzed in order toand
uncompetitive extract
chemicalmixedchemical inhibition
reac- requiring cell processes. Its role in the cell is analogous to that of
V0 ! "" (629) are observed only foraenzymes with two or more sub-
Km # !$[S] tionsenergy in cells and are convert
stratessay,
functionally it into form useful
organized
S2and are veryActivation
S1 andfree-energy-yielding
into to the many
important
cell; se-
barrier
to-
in the
money in an economy: CO2 it is earned/produced in exergonic reactions
quences gether ofthese consecutive degradative, reactions, called pathways,
(transition reactions
state,in ) and spent/consumed in endergonic ones.
NH
experimental analysis of such Aenzymes.
medical Iftherapy
an inhibitor based on competition at the ac- the 3 eyes are particularly sensitive to formaldehyde.
Free energy, G

which arethe designated

product
binds toof
catabolism.
theone sitereaction
Other becomes pathways bythe
start with
reactant
[I] [ES][I]
normally occupied tive site Sis1,used
it may toact
treat as patients who have ingested Si invariably HEthanol
2 Orequire the competes
rs effectively with methanol as an alter-
small precursor molecules and convert them to pro- which mp so input of energy, are col-

!G
! 1 # "" and K$I ! "" in the next. aSomecompetitive pathways inhibitor degrade
in experimentsorganic innutrients
which [S ] is
cur
uncat
methanol, a solvent found !G1in
cat gas-line antifreeze. The liver ledesignated
produnative presubstrate for alcohol dehydrogenase. The effect of
Reactants (A)
K$I [ESI] lectively cts,anabolism. The overall network of
gressively
into simple end products in order larger
varied. If and
an more
inhibitor complex
binds
enzyme to molecules,
the
to extract site
alcohol chemicalnormally includ- occu-
dehydrogenase converts methanol to
enzyme-catalyzed ethanolpathways is much constituteslike that of a me-
cellular competitive inhibitor, with
by Equation 629, at high concentrations ing proteins
energy and convert it into a form pied and by Snucleic
2, it mayacids. act as Such a mixed synthetic
useful to the
formaldehyde, or uncompetitive
which pathways,
cell;is to- in-
damaging to many tissues.tabolism. Blind- ATP is the thedistinction
major connecting link (the shared
that ethanol is also a substrate for alcohol
V0 approaches Vmax/!$. Thus, an uncom- hibitor of S1. The actual inhibition patterns observed
!G
FIGURE central roleCO
128 Theintermediate) of 2ATP in
between themetabolism.
catabolic andATP is thecom-
anabolic
ness is a common result Products
of methanol (B) ingestion,
gether thesedepend
itor lowers the measured Vmax. Apparent degradative,
on whether free-energy-yielding
the S1- and S2-binding reactions
events are shared chemical because
intermediate
ponents NH
dehydrogenase
linking
of this
and toits
energy-releasing
network
3 (shown schematically
concentration
energy-in Fig.
will decrease over
eases, because the [S] required are designated
to reach catabolism.
ordered or random, Other
and thus pathways
the orderstart in which withsub- requiring cell processes. 128). The pathways
Its role in theofcell
enzyme-catalyzed
is analogous reactions
to that of that
S H2O s
decreases by the factor !$. small precursorstratesmolecules and convert leave thethem
Reaction coordinate (A
activeto sitepro-
B)
actimonitplisthe main constituents or of cellsproteins, fats,
bind and products can be money in an economy: earned/produced ursin exergonic reactions
inhibitor (Fig. 615c) also bindsgressively
at a site determined.
larger Activation
and(transition
more barrier
Use ofcomplex
one
FIGUREof 127
themolecules,
reaction
Energy changesproducts
during aaschemical
includ- an reaction. An acti- sugars, eand pronucleic prec
ducts,ones.
acidsare virtually identical in all
state,barrier, and
) representing the transition state, must be overcome in spent/consumed in endergonic
the substrate active site, but it binds to ei- vation living organisms.
ing proteins inhibitor
and nucleic is often particularly
acids. Such informative.
synthetic If only one of
pathways,
Free energy, G

The rate equation describing mixed inhi- the conversion of reactants (A) into products (B), even though the prod-
two reaction products is present, no reverse reaction FIGURE 128 The central role of ATP in metabolism. ATP is the
ucts are more stable than the reactants, as indicated by a large, neg- Metabolism Is Regulated to Achieve Balance
can take place. However, a product !Ggenerally
binds to
(!G). The energy requiredshared
whichchemical invariably require the input of energy, are col-
intermediate
Reactants
some
ative free-energy
(A) active
part of the site,
!Gthus
changeuncat to overcome the and Economy linking energy-releasing to energy-
cat serving as an energy
inhibitor. lectively designated anabolism.
role in the Thecell isoverall network
to thatofof

Vmax [S] activation barrier is the activation (!G ). Enzymes catalyze
requiring re-
cell processes. Its analogous
V0 ! "" (630) Enzymologists can use elaborate kinetic studiesbarrier.
involv-
They bindenzyme-catalyzed Not only do living cells simultaneously synthesize
me-thou-
itpathways constitutes cellular
!Km # !$[S] actions by lowering the activation the transition-
money in an economy: is earned/produced in exergonic reactions
ing different combinations
Activation barrier and amounts of products and
state intermediates tightly, and the binding energy of this interaction sands of different kinds of carbohydrate, fat, protein,
!$ are defined as above. A mixed inhibitor inhibitors to develop a detailed picture of!Gthe mecha- and tabolism.
spent
ATPand
/consumed is the
in major
endergonic
nucleic acid connecting
ones.
molecules and link (the
their shared
simpler subunits,
(transition state, )
effectively reduces the activation energy from !G uncat to !G cat . (Note
Products
energy(B) intermediate) between
but they dothe so catabolic
in the preciseandproportions
anabolicrequired
com- by
Free energy, G

s both Km and Vmax. The special case of nism of a bisubstratethat reaction.

activation is not related to free-energy change, !G.)
y encountered in experiments, classically ponents of this network (shown schematically in Fig.
ned as noncompetitive inhibition. Ex- Inhibition The irreversible
Irreversible(A) !Guncat inhibitors are which 128).invariably
The pathways require the input of energy,
of enzyme-catalyzed reactionsarethatcol-
Reactants
on 630 to see why a noncompetitive in- Reaction coordinate
!Gcat (A B)
those that bind covalently with or destroy a functional lectively act on the designated anabolism.ofThe
main constituents overall network
cellsproteins, fats,of
affect the Vmax but not the Km. group
FIGURE 127 on anchanges
Energy enzymeduring
that ais chemical
essentialreaction.
for the enzymes
An acti-
enzyme-catalyzed
sugars, and nucleicpathways acidsareconstitutes cellularin me-
virtually identical all
630 serves as a general expression for the activity, or those that form a particularly stable nonco- tabolism. ATP is the major connecting link (the shared
living organisms.