Introducción a la

Cinética Enzimática

Prof. Pablo Araujo Granda Ph.D.

paaraujo@uce.edu.ec
Lecturas indispensables

• Cap.8 Enzimas conceptos básicos y cinética

- Berg Tymoczko Stryer Bioquimica 2008

• Cap11. Enzimas Catalizadores Biológicos -

Mathews 2002
¿cuánto sabes de las Reacciones Químicas?
• Igualación de ecuaciones químicas
• Química General 1

• Estequiometría y Equilibrio Químico

• Química General 2

• Balances de materiales con y sin Rx. Química.

• Cálculos Básicos 1 y 2

• Energía involucrada con las Rx. Químicas

• Termodinámica
entually
of time at take ical yield.
a product When
rather thatreactants
reactantorisproducts
consumed areininvolved in additional
other reactions;
Consider the general irreversible re
is the rate at the reactions,
observedthe observed
yield yield would
of product may bebedifferent~om
lower than thethe theoretical
theoret-
ortant char-
ntually yield.
takeEstequiometría
ical yield. When reactants or–products
Rendimiento
are involved in additional
ny
is thedifferent The above
rate at reactions, the analysis leads may
observed yield to two useful definitions
be different~om of yield for
the theoretical
tion ofchar-
ortant reac- yield.
reaction systems:
nye properties a A + bB ---> y Y + z Z .
different The above analysis leads to two(total useful definitions
mass of yield for
or moles of)
chapter.
tion of reac- reaction systems:
/true, stoichiometric or] = product formed
e properties k theoreticalyield J (mass (totalor moles
mass of reactant
or moles of) used
chapter. /true, stoichiometric or] = kt~ form particular product ]
thatformed
product
k theoreticalyield J (mass or moles of reactant used (11.9)
products is kt~ form that particular product ]
The rate of this reaction can be repre
, yield is the and
products
(11.9)
amount isof observed or ~ (massor moles of product present )
, there sion of compound A; let us use the s
yield isisthe
no
and
apparentoryield]
amount are
rameters of observed ~ = (mass
( t~176176176
or moles)ofc product
o n s u mpresent
ed )
dthere ofreaction with respecttoA. RA has u
is no
to express
rameters are apparent yield] = ( t~176176176 ) c o n s u m e d (11.10)
l acceptance
d to express
How do we measure reaction r
repared for There is a third type of yield applicable in certain situations.
(11.10)
s fall into this cat-mined exclusively by the thermody
ions
the rateand
of
take place in the
tion is executed. Equilibrium concentration
or under- reactants
Consider a
equilibrium and products;
reversible reaction
constant, K Forit isreaction
indepen
represented
the of
oadients.
this cat-Analysis
equation:
complexes,
cation of mass-
Equilibrio
tion is executed. Químico
Equilibrium concen
ce in the cygcz z
s;reaction
the lattertheory.equilibrium constant, K For the reac
Analysis
n-growing.
er 12.
A+ K ...
bB ~ y Y +
CAe CBeb
z Z .
of
rigin. mass-
Celltheory
reaction cygcz z
he catalyst
theory.
speed of homo- where ... CAe,
A, B, KY and Z areCse, Cye and
chemical Cze are
species; equilibri
b, yand za
alytic
factorsreac-
affecting of A, B,CAe CBeb
Y and Z, respectively. The value of
coefficients. If the components are left in a clo
on rate and perature as follows:
n theory
• K =be
infinite period of time, the reaction proceeds
f(T) ……. (Depende de la Temperatura entre otras cosas)
s must
of namic
homo- where CAe,- ACse,equilibrium is reached. At equilibrium
G ~r x n Cye and Cze are eq
eactors.
driving force
I n K = for further change; the reaction
affecting limit
distinction of A, B, Y and R T
Z,
of its capacity for respectively. The va
chemical transforma
eaction thermody-
actions. A
rmodynamics system.is Composition of the equilibrium m
11.1.3 Reaction Rate
The rate of this reaction can be represented by th
sion
Consider of compound
the A;
general let us use the symbol
irreversible RAret
Cálculos
ofreactionBásicos
with respect–toA.
Balances
RA has units of, for e
How do we measure reaction rates? For a
asystem,
A + rate
bB of reaction
---> y Y + z Z .to rate of ch
is related
the system by the unsteady-state mass-ba
derivedGeneral
Ecuación in Chapter 6: de masa:
de Balance

dM
The rate of
- this reactiona :can
be repre
.
dt
sion of compound A; let us use the
ofreaction with respecttoA. RA has u
 ts the rate of
occurringConsiderin solution,a otherreaction
reversible standard condit
represented
m or under- .Acygcz z
oequation:
[1] Grin is equal– to
Termodinámica
complexes,
K ... the difference
Energía de Gibbs in stand
CAe
formation,
es; the latter CBebG ~, between products and reactan
on-growing. A+ bB ~ y Y + z Z .
most stable form at A G= A H - TAX
origin.
whereCell
hemical ACAe,o Cse, Cyeoand Cze. are equilibrium
reactions o_ conce
the catalyst Grx n = y Gy + z G Z - G A b G~ .
of A,
ions mayB, Ybeand
A, Z,
used B, Y respectively. The value
and Z are chemical of Kdepends
species; b, yand z
atalytic reac-
eionperature
form
dardj~ee at
rate and
as follows:
energy oAf G= Therefore,
coefficients. A IfHthe
- from Eq. (11.3):
TAX
components are left in a cl
nts:
reactions infinite period of time, the reaction procee
ers must be - A Gfree
Standard ~r x n energies ofisformation are availa
namic equilibrium - A reached.
H ~ x n At equilibriu
AX~x n
yreactors.
be such
used
I n K =as those
driving listedIn in
force K=
forSection
~ - change;
further 2.6.
+ the reactio
distinction RT RT R
energy Free Therefore,
o f (11.4)
limit
energy from
of itsGcapacity
is Eq.
related (11.3):
for chemical
to transform
enthalpy H
reactions. A
system. Composition of the equilibrium
absolute temperature Tas follows:
l concentra-
Termodinámica – Energía de Activación
 … es el momento de
aprender algo mas …
Velocidad de reacción => ¿cómo cambia la concentración de
reactivo ó un producto en función del tiempo? => Cinética
 kg m -3 s-1. Rate of reactio
basis is used to account for d
Velocidad de unareaction
reacción química
systems. Therefore
closed system has volume V
• Masa del reactivo (sustrato)
convertido es proporcional
tamaño del sistema
RA _ - 1 d M A
~A m
(volumen) V V dt
• Masa*volume-1*tiempo-1
oring the change - d q
• Moles*volume-1*tiempo-1 ~A m
s of reaction rate where rA is thedtvolumetric r
• V = cte
a can be analysed A. When V is constant, Eq.
 (11.12). Often but not always,
Consider the general irreversible re
tion can be expressed
Cinética de reacción
as a funct
using the following mathematic
a A + bB ---> y Y + z Z .
r =kqq
k = constante de velocidad (Coeficiente de velocidad)
The rate of this reaction can be repre
k = Independiente de la concentración / dependiente
de la temperatura
sion of compound
where A; let
k is the rate us use or
constant theras
CA / CB = Concentraciones de los reac?vos
ofreaction with respect
definition,
estequiometría)
toA.
a / b = Orden de reacción (nada que ver con la
the rate RA hasisu
constant
Howtion do we measure
of reacting reaction
species but is der
 er volumetric
yst of low specific
t a higher cell Temperature
where k is the rate has
constan a
catalyst concentra-
Temperatura vs. k
Variation
quencyfactor, of
Eis the
the rate
activ
nt of a more
roductivity will be
ideal gas constant, and Ti
red • to
of
achieve
organism
Ecuación de
the
Arrhenius by the Arrhenius equ
symbol R A will be are listed in Table 2.5 (p
• A = constante de Arrhenius (factor de frecuencia)
ates.
pect to (factor
component equation, as Tincreases, k
pre-exponencial) E
cesrate.are inter-
• E = energía
natural
de activación
k = A e -
logarithm /RT
of both
• R = constante universal de los gases
e, high total
• T = temperatura absoluta In k - In A -
E
f low specific RT
 11.3.1 Zero-Order Kinetic
Reacciones de Orden Cero
Ifa reaction obeys zero-order kinetic
a rpendent
an equationof for
= independiente
A de la CA as concentration.
a function
concentración
reactant of tim
de reactivo
te data can then be checked directly against
n- tion. Integrating Eq. (11.24) with initial
ly
r =k0
at t - 0 gives:
h-
2. CA= f-rA dt = CAo- kot.
where ra is the volumetric rate of rea
be
n. k o is the zero-order rate constant, k o a
ationship
1 1 . 3against
. 2 F i rthe
st-O r d e r Kequation.
i n e t i c s Separ
resulting
rA - -dCA/dt, and then check the measur
follows:
If a grating
Reacciones
reactionEq. de(11.27)
obeysprimer with
orden
first-order initial cond
kinetics,
against the resulting
gives: equation. Separati
between reaction rate and reactant concentra
grating Eq. a(11.27)
r = depende
A withde initial
la concentración reactivo conditi
(11 9 CA A=CAo e-k't
gives: r A = A1C
the first-
CA =CAo
ero-order e-k't
Taking natural logarithms of both side
where rA is the volumetric rate of reaction a
ds on the
order rate constant with dimensions T -1. Lik
constant In CA previous
of the = In CA0- k1 t. the value of k
section,
reaction
Taking natural
catalyst logarithms of both sides:
concentration.
 Lactate
where dehydrogenase
CA has units gTable 11.3. Km and
1-1 and t has Bacillus
units o
h. Th
sonably well rep-
Penicillinase Bacillus
Aplicando
source of the enzyme.
a las enzimas tenemos:Jack be
Urease
If we adoptKinetic
11.3.3 Michaelis-Menten conven
The kinetics k2
and call reactant A the
E + S of mostESenzyme
--o reactions
E+P are rea
resented by thek_l in the familiar form:
Michaelis-Menten equation:

(11.30)
Vma CA Vma x $
where E is enzyme, SVis msubstrate and P is p
~A
Km+s
Km+q complex. Binding of substra
enzyme-substrate
is the inconcentra-
the first step is considered reversible with
ate of constant
reactionk1at and reverse reaction constant k_l
Cinética Enzimática – Saturación Sustrato
is considered reversible with forward reaction tration; in this concentration range, the reaction is essentially
reverse reaction constant k_l. Decomposition zero orderwith respect to substrate. On the other hand, at low
ubstrate complex to give the product is an irre- substrate concentrations s << Km, the value of s in the denomi-
n with rate constant k2; k2 is known as the nator of Eq. (11.31) is negligible compared with Km, and Eq.
. Analysis of this reaction sequence yields the (11.31) can be simplified to:
Cinética mixta - 11.5Dos
Figure opciones
Michaelis-Menten plot.

(11.33) V

Vmax
oncentration of active enzyme. As expected in
ns, enzyme E is recovered at the end of the
• Catalizador (enzima) Zero-order
feature of Michaelis-Menten kinetics is that region
se satura a elevadas
omes saturated at high substrate concentra-
.5 shows the form of Eq. (11.31); reaction rate
ease indefinitely with substrate concentration
concentraciones de
a limit, Vmax. At high substrate concentrations I
the denominator of Eq. (11.31) is negligibly
sustrato
with s so we can write:

I region

(11.34)
 romyces cerevisiae If we adopt Sucrose
convention
v does not increase i
and call reactant A the sub
pora crassa but approaches Sucrose
a
in the familiar form: limi
Cinética
s subtilis mixta – Dos opciones
s >> Km, Km Lactatein the d
(11.30)
s licheniformis small compared
V m Benzylp
with
Vma x $

Km+s
ean
reaction,• Opción
CA is the
1: concentra- Urea
maximum rate of reaction at
• S >>> KM Urnax $
Km is the Michaelis constant where v is the volumetric r
• KM se
dimensions Km has the o r concentration. The biochem
desprecia
as rA;
pical units for Vmax are equation will not be cov
• Orden cero
~
or Km are k g m o l m - 3 . As reaction ~n.lax 9
models and assu
(11.32) to Eq. (11.31) can be found in
lumetric rate proportional
sent. The Michaelis constant
 source of the enzyme.
equation:
If we adopt conventiona
and call reactant A the subs
Cinética mixta – II
Dos the opciones
in Homogeneous
familiar form:Reactio

(11.30) Vma x $
V m
• Opción 2:
eaction, CA is the concentra-
Km+s
• S <<< KM
maximum rate of reaction at
• SMichaelis
Km is the en denominador
constant where v is the volumetric ra
Vmax
imensionsseas desprecia
rA; Km has the concentration. The biochem
ical units for Vmax are
• Primer orden
r Km are k g m o l m - 3 . As
Km
equation will not be cove
reaction models and assum
umetric rate proportional to Eq. (11.31) can be found in
¿como obtener valores de Vmax y KM?

• Origen = Reactor discontinuo

• Varios ensayos a diferentes concentraciones de sustrato

• Datos de velocidad inicial (tiempo cero)

• Datos de rA en función de S

• Gráficos:
• Michaelis-Menten / Lineweaver-Burk / Eadie-Hofstee / Lagmuir
 that the enzyme concentration is known, it is advantageous to define a
Kinetics
quantity k , the 'catalytic constant' K
cat or m values
'turnover forassome
number,'' V/e . Forenzyme-
the 0
Michaelis-Menten mechanism, kcat isTable
identical with but
k + 2, K
11.3. m inand
generalother
the enz
s aremore non-committal
reasonably Michaelis-Menten
notation
well (1913)
rep-kcat is preferable.
source of the enzyme.
uation:
If we adopt conventional s
and call reactant A the substrat
in the familiar form:

(11.30) Vma x $
V m

tion, CA is the concentra-

Km+s
imum rate of reaction at
m is the Michaelis constant where v is the volumetric rate o
ensions as rA; Km has the concentration. The biochemica
Figure 2.1 Plot of initial velocity, v, against substrate concentration, s,for a reaction obeying the
 source of the enzyme.
If we adopt conventional symbols for biological
INTRODUCTION TO ENZYME KINETICS
and call reactant A the substrate, Eq. (11.30) can be r
Michaelis-Menten
log s. This plot gives
in athe
symmetrical
familiar S-shaped (1913)
form: curve, as shown in Figure 23,

(11.30) Vma x $ Log (s//CJ

V m

is the concentra-
Km+s Figure 2.3 Determination ofW and Km by the method of Michaelis and Men
straight over an appreciable range of velocities, and so the maximum slope
easily
ate of reaction at
which has maximum slope when s Km. Differentiation
Michaelis constant 0.51/ where v is the volumetric
shows that rate of reaction and s is the s
as rA; Km has the concentration. The biochemical basis di; of theKmVs
Michaelis-
for Vmax are dins (Km + s)2
equation will ornot be covered here; discussion of
k g m o l m - 3 . As reaction models and assumptions dvinvolved 2.303 Kmin
Vs deriv
e proportional to dlogs (Km + s)2
Eq. (11.31) can be found in biochemistry texts [2, 3]. S
The maximum slope occurs when s = Km9 and is equa
ichaelis constant to say here that0.576V.
the simplest
Hence, V canreaction
be estimatedsequence which
by dividing the maximua
which rA = Vmax/2. Michaelis and Menten then estimated Km as the value of s w
Loghalf
for the kinetic was (s//CJ
properties of can
of V. This many enzymes
be achieved is:
more accurately than e
at which the maximum slope occurs. This plot is not usuall
it is of interest for several reasons: it emphasizes the relation
 uni
plo
1 1.4.4 Langmuir Plot
Langmuir (1918) / Hanes (1932) sec
Multiplying Eq. (11.37) by s produces the linearised form of mi
the Michaelis-Menten equation according to Langmuir: for
vid
s -- Km + s val
v Vma~ Vma~ tio
(11.39) det
kn
Therefore, a Langmuirplot of s/v versus s should give a straight
line with slope l/ Vmax and intercept Km/Vmax" Linearisation of 11
data for the Langmuir plot minimises distortions in experi-
mental error. Accordingly, its use for evaluation of Vmax and En
Km is recommended [6]. can
enz
 line plot from which Vm~ and Km can
I I I I Inverting Eq. ( 11.31) gives:
8 9 10
Lineweaver-Burk (1934)
11 1

1 Km 1
-- +

v Vmax s Vmax
yme kinetics, as illustrat-
e is maximum at some
pH is moved either side
so that a pl0t o f 1/v versus 1/s should give a
ons have been developed
slope Km/vm~ and intercept l/vmax. This
me activity; however, the
plot is known as the Lineweaver-Burk plot,
d experimentally. Ionic
found in the literature on enzyme kinetics.
onsiderable influence on
earisation process used in this metho
elations are available for
experimental error in v (see Section 3.3.4) so
are amplified at low substrate concentrati
quence, the Lineweaver-Burk plot often
 I I I I I Inverting Eq. ( 11.31) IfEq. (11.37) is mul
gives:
ng 7 kinetic
8 9parameters
10 11 for 1 Michaelis-Menten
and then reac-
rearranged, an
ofsubstrate con-
not as straightforward as for zero- and first-order
pH
mined
. SeveralGráfico
from
graphical batch
methods are Michaelis-Menten
1
available;
--
K munfortu- 1
+
equation
me do Eadie-Hofstee
not give accurate results. v Vmax s Vmax
11.2.
enzyme
irst step inTypically,
kinetics, as illustrat-
kinetic analysis of enzyme reactions is to
rate is maximum at some and then rearrang
fhat
ata for
the pH several
is moved batch
rate of reaction v as a
either side
function v
ofsubstrate Vmaxcon- v
n s. Rates of reaction can be determinedso that a pl0t
u

fromo fbatch m
Michaelis-Menten
1/v versus 1/s should give a e
ial
uations substrate
ation data as described con-in Section
have been developed
s
slope 11.2. Km
Typically,
Km/vm~ Km
and intercept l/vmax. This
zyme activity; however, the
ial
nmined rate data are used. This means
rateexperimentally.
is evaluated plotthat
is several asbatch
known the v
Lineweaver-BurkVmax plot,v
Ionic u m

ents are carried out with differentfound initialinsubstrate con- on enzyme

ve considerable influence on
ns; from each set of data the reaction
orrelations are available for
me Kinetic
ata
the literature s Kmkinetics.Km
rate is evaluated
earisation process used in this meth
experimental error in v (see Section 3.3.4) s
are amplified at low substrate concentrati
quence, the Lineweaver-Burk plot often
results and is therefore not recommended [3
 disadvantage that v, usually regarded as the depen
both co-ordinates. On balance, the plot of s/v agains
Eisenthal and
of the three.
INTRODUCTION TO ENZYME KINETICS
Cornish-Bowden (1974)
Eisenthal and Cornish -Bowden (1974) have recent
different method of plotting enzyme kinetic results,
linear plot. Instead of writing the Michaelis-Mente
way to show the dependence of v on s, they rearran
dence of V on Km:

V = v + -sKm

Thus, for any values of s and t;, it is possible to plot V

line with slope v/s, intercept — s on the Km axis, and
This straight line relates all values of V and Km th
values of s and v exactly. If straight lines are draw
observations, they should intersect at a common p
which give the only values of V and Km that satisfy a
Figure 2.7 Direct linear plot o/V against K : Each line represents one observation, and is drawn
m
 the volume of the fermenter?
(ii) The cell concentration is 20g1-1 dry weight.
Calculate the specific productivity.

Ejercicio
11.4 Enzyme kinetics
actions Lactase, also known as ~galactosidase, catalyses the hydrolysis
of lactose to produce
I I HomogeneousReactions glucose and galactose from milk and
whey. Experiments are carried out to determine the kinetic
parameters for the enzyme. Initial rate data are listed below.

Lactose concentration Initial reaction velocity 11.6 Enzyme reaction and deactivation
(mol l- 1 X 10 2) (mol 1-1 min- 1 X 103) Lipase is being investigated as an additive to laundry de
for removal of stains from fabric. The general reaction
2.50 1.94
2.27 1.91 fats ~ fatty acids + glycerol.
1.84 1.85
1.35 1.80 The Michaelis constant for pancreatic lipase ig 5 m
1.25 1.78 60~ lipase is subject to deactivation with a half-life o
0.730 1.46 Fat hydrolysis is carried out in a well-mixed batch
0.460 1.17 which simulates a top-loading washing machine. The
0.204 0.779 fat concentration is 45 gmol m -3. At the beginning
reaction the rate of hydrolysis is 0.07 mmol 1-1 s-1. Ho
Evaluate Vmax and Km. does it take for the enzyme to hydrolyse 80% of the fat p

11.5 Effect of temperature on hydrolysis of 11.7 Growth parameters for recombinant

starch
 83 mM Sucrose
time (min) [P]/[S0] Const
time (min) [P]/[S0] Const
49.5 0.352 0.0482
49.5 0.352 0.0482
90.0 0.575 0.0447
90.0 0.575 0.0447
Current
Current Topic
125.0 Topic
Current 0.690 0.0460
125.0 Topic 0.690 0.0460
m
m of
of the
the Ejercicio
equation
equation
This
m of the equation
terms
terms F/K
terms F/K and
F/K and G/
and G/
G/
FFF
for
for con Datos
Table
differential
Thisfor de MM
1.
equation
Table
differential (1913)
Michaelis✓Menten
1. Michaelis✓Menten
equationwas
wasthen
then integrated
Michaelis✓Menten Global
integrated Global
to
to yield
Global Data Fitting
yield Data
Data Fitting
151.0
151.0
a
aa
Fitting208.0
208.0
0.766
0.766
0.900
0.900
41.6mM
mMSucrose
Sucrose
0.0456
0.0456
0.0486
0.0486
333
333 mM
mM Sucrose
Sucrose 41.6
product inhibition.
product inhibition. 333 mM Sucrose
product inhibition. time (min)
time (min) [P]/[S
[P]/[S 0] 0
] Const
Const
ibition had
bition had not
not yet
yet time
time (min)
(min) [P]/[S
[P]/[S000]]]
[P]/[S Const
Const
Const
ibition had not yet 10.25
10.25 0.1147
0.1147 0.0406
0.0406
ted
ted here mathemati-
ed here
here mathemati-
mathemati- 77 0.0164
0.0164
0.0164 0.0496
0.0496
30.750.0496
30.75 0.3722
0.3722 0.0489
0.0489
14
14 0.0316
0.0316
0.0316 0.0479
61.750.0479
0.0479 0.615 0.0467
61.75 0.615 0.0467
their postulate
their
their postulate was
postulate was
was 26
26 0.0528
0.0528
0.0528 0.0432
90.75
90.75 0.0432
0.0432 0.747
0.747 0.0438
0.0438
ull time
ull time dependence
dependence 49
49 0.0923
0.0923
0.0923 0.0412
112.70
112.70 0.0412
0.0412 0.850
0.850 0.0465
0.0465
trations to
trations to derive
derive aa 75
75 0.1404
0.1404
0.1404 0.0408
132.70
132.70 0.0408
0.0408 0.925
0.925 0.0443
0.0443
values
alues of of KKSS,, KKFF,, and
and 117
117 0.2137
0.2137
0.2137 154.700.0407
154.700.0407
0.0407 0.940
0.940 0.0405
0.0405
values S F
uires including
ires including This mass
Thisequation
equationallowed
mass allowed
1052 theconstant
the
1052 constantterm (Const0.9834
term (Const == C/KS) to be
0.9834
0.9834 [0.0498]
[0.0498]
[0.0498] 20.8
20.8mMmMSucrose
Sucrose
a form
form with
with calculated
aa single frommeasurements
singlefrom
calculated measurementsofofthe 166.7
166.7 mM Sucrose
mM
the concentration
concentration of product
Sucrose time
time (min)
(min) [P]/[S 0] 0]
[P]/[S Const
Const
form with a single
G. (F)asasa afunction
(F) functiontime
ofoftime
time time (t)atatvarious
(t)
(min) various starting
starting [P]/[S
concentrations
concentrations
G. (min) [P]/[S000]] 17 Const
17 Const
Const 0.331
0.331 0.0510
0.0510
of sucrose, S . Michaelis and Menten converted their optical
of sucrose, S0. 0 Michaelis and Menten converted0.0350 their optical 27
27 0.0444 0.452
0.452 0.0464
0.0464
rotation data to obtain 88 the fraction of product formed 0.0350 relative 0.0444
0.0444
rotation data to obtain16the fraction of product formed 0.0636 relative 38
38 0.0446 0.611
0.611 0.0500
0.0500
16 as0.0636 0.0446
0.0446
totostarting
startingsubstrate
substrateconcentration,
concentration, [P]/[S
28
[P]/[S0],], as
0
illustrated in
illustrated
0.1080 in 62
62 0.0437
0.736
0.736 0.0419
0.0419
Table1.1.They
Theyshowed
showed 28 that,indeed,
indeed, thethe constant
constant term, C/K
0.1080 C/KS,, 95 0.0437
0.0437 0.860 [0.0388]
Table 52 that, term,
0.1980 S 95 0.0444 0.860 [0.0388]
was “verysimilar
was“very similarininallall
52 experiments and despite small
experiments and despite small variation
0.1980variation 0.0444
1372 0.0444 0.990 [0.058]
82 0.3000 Biochemistry
1372 0.0445 0.990 [0.058]
showsnonotendency
shows tendencyfor for
82 systematic deviation neither
systematic deviation neither 0.3000 with time
with time
a
aConst mean
0.0445
value = 0.0454 min✓1✓1
0.0445 . This reproduces the data from the
103
nor with sugar concentration,
103 so that we can 0.3780
conclude that we Const mean
0.0454 value = 0.0454 min . This reproduces the
0.0454 table in ref 1. Michaelis and Menten analyzed
data
prior from the substrate
to significant
nor with sugar concentration, so that we can83conclude 0.3780 that we last (unnumbered)
0.0454 these
can conclude that the value is reliably constant.” mM Sucrose last (unnumbered) table in ref 1. Michaelis and Mentenand analyzed
Km forthese
83 mM Sucrose data using the integrated form of the rate equations to compute asubstrate turn
can conclude that the value is reliably constant.” data using the (Const = C/KS), as described in the text. We fit these a
integrated form of the rate equations to compute
inhibition. Lineweaver and
This extraordinary time analysis allowed fitting of[P]/[S
(min) the 0full
] time single constant
Const
This extraordinarytime analysis
(min) allowed fitting of[P]/[S
course of product formation to the integrated form of00 the rate
the full
] time single constant
Const
Const (Const = C/K
data globally on the basis of numericalS ), as described in the text. We
analysis
integration of the rate fit these
to parse the kin
equations
course of product 49.5
formation to the integrated form0.352of the rate data globally
0.0482
to give the0.0482 on the basis of numerical
results shown in Figure 1. integration of the rate equations
reciprocal plot. This type of
equation to extract a49.5 single unknown constant that0.352 accounts for to give
0.0482
the results shown in Figure 1.
equation
all of to theextract
data.a single
90.0 unknown constant that0.575
90.0
In doing so, Michaelis and accounts
0.575 Menten for 0.0447
0.0447
0.0447
most of the 20th Century. A
alldemonstrated
of the data. that In
the doing
125.0
125.0 variationso,in Michaelis
the rate of and 0.690 Menten
0.690
turnover as a Their data fitting provided an average C/KS value rate
0.0460
0.0460
0.0460
equations
of 0.0454 ± (also know
Their data fitting the ±
demonstrated
function of that timethe
151.0
and
151.0 variation
substratein the rate of turnover
concentration 0.766
0.766 could asbea 0.0032 min ✓1
0.0456
0.0456
0.0456
✓1
, fromprovided
which weancanaverage C/K
calculate S value
Vmax of 0.0454
= keliminated
catE0 = 0.76
need for s
0.0032 min , from which we their
can calculate VKmax quantitative
= kcatofE0 16.7 analysis
= 0.76 of full
grated to yield
function
rated to yield of time
understood as a constant and
208.0 substrate concentration 0.900 could be ± 0.05 mM/min
0.0486 based upon reported value
208.0 defining the rate of product formation S 10

understood as a constant defining the rate of product

41.6 mM
0.900
formation
Sucrose
0.0486
0.0486
± 0.05 mM/min
mM. based upon their reported KSCarl value Frieden. One can
of 16.7
based upon the calculated concentration 41.6 of the ES
mM Sucrosecomplex. parameters and product inh
based mM.
This upon the calculated
(min) concentration of itthe ES 0complex.
This
is a remarkable
thatisthe
a remarkable
time contribution.
time
constant derived
that the constant derived
(min)
contribution.
10.25
However,
However,
by Michaelis
should
anditMenten
10.25 by Michaelis and Menten
[P]/[S
[P]/[S
should be
0.1147
be
0]
†
] noted Figure 1. Global fitConst
COMPUTERto the data of Michaelis and Menten. The data
Const
Const ANALYSIS
noted from Michaelis and Menten (reproduced in Table 1) were fit by
in fitting
0
COMPUTER
0.0406
0.1147in fitting simulation using KinTek
0.0406
0.0406 9ANALYSIS
Explorer with the only variable being k E
course data directly using co
laborious initial rate analysi
early work in enzymology t
Homework

• Ejercicios del 1 al 15 (todos) Stryer 2008

• Ejercicios del 1 al 27 (todos) Mathews 2002