ENZIMAS:

GENERALIDADES
186 Enzymes

where E, S, and P represent the enzyme, s

FIGURE 6–1 Binding of a substrate to an enzyme at the active site.
The enzyme chymotrypsin, with bound substrate in red (PDB ID
7GCH). Some key active-site amino acid residues appear as a red
product; ES and EP are transient complex
zyme with the substrate and with the prod
To understand catalysis, we must firs
Noé Rigoberto Rivera. MD
the important distinction between reacti
Biología Molecular Ph.D
and reaction rates. The function of a cata
crease the rate of a reaction. Catalysts d
Departamento de Bioquímica
reaction equilibria. Any reaction, such as
Facultad de Medicina
described by a reaction coordinate diagram
pictureUniversidad de El Salvador
of the energy changes during the rea
cussed in Chapter 1, energy in biological s
scribed in terms of free energy, G. In th
diagram, the free energy of the system is pl
the progress of the reaction (the reaction
The starting point for either the forward o
reaction is called the ground state, the
to the free energy of the system by an aver
(S or P) under a given set of conditions.
 Enzimas

Polímeros biológicos que catalizan las

reacciones químicas que hacen posible la vida

Casi todas las enzimas son proteínas, excepto

las robozimas (RNA)
 Enzimas
Incrementan el índice de reacción en el orden
del 106

No se consumen durante la reacción

Especificidad absoluta o relativa: tipo de

reacción y sustrato
 istic: they are self-splicing—no protein enzymes are in- These reactions are very similar to the DNA breaking and
volved. Group I introns are found in some nuclear, rejoining reactions promoted by topoisomerases (see Fig.
mitochondrial, and chloroplast genes that code for 24–21) and site-specific recombinases (see Fig. 25–40).
rRNAs, mRNAs, and tRNAs. Group II introns are gener- The group I splicing reaction requires a guanine nu-

La mayoría de enzimas son proteínas

ally found in the primary transcripts of mitochondrial or cleoside or nucleotide cofactor, but the cofactor is not
chloroplast mRNAs in fungi, algae, and plants. Group I used as a source of energy; instead, the 3!-hydroxyl
and group II introns are also found among the rare exam- group of guanosine is used as a nucleophile in the first
ples of introns in bacteria. Neither class requires a high- step of the splicing pathway. The guanosine 3!-hydroxyl
energy cofactor (such as ATP) for splicing. The splicing group forms a normal 3!,5!-phosphodiester bond with
mechanisms in both groups involve two transesterifica- the 5! end of the intron (Fig. 26–15). The 3! hydroxyl
Enzimas: tion reaction steps (Fig. 26–14), in which a ribose 2!- or Ribozimas: RNA
of the exon that is displaced in this step then acts as a
Proteínas, estructura primaria, Intron
86 Enzymes
secundaria, terciaria y cuaternaria:
conformación nativa Primary
transcript
where E, S, and P represent the enzyme, sub
5 ! Exon 3 ! Exon
5! product; UpA ES and EP Gare pU transient complexe 3!

zyme with the substrate The 3and

! OH ofwith the produc
guanosine
acts as a nucleophile,
pG OH
To understand catalysis, we must
attacking the phosphate
the 5 ! splice site.
at first
the important distinction between reactio
and reaction pGpA rates. The function of a cataly
crease the rate of a reaction. Catalysts do
Intermediate

reaction equilibria.
U OH
Any reaction, such as S
GpU
5! 3!
described by a reaction coordinate diagram (F
The 3 ! OH of the 5 !exon
picture of the energy changesbecomes the during
nucleophile,the reac
completing the reaction.
cussed in Chapter 1, energy in biological sys
scribed in terms of free 5 ! pGpA
energy, G. In the
FIGURE 26–15 Splicing mechanism of group
diagram, the free energy of the system G OHis 3 ! plo
Spliced RNA
I introns. The nucleophile in the first step may the progress of the reaction (the reaction c
be guanosine, GMP, GDP, or GTP. The spliced intron
is eventually degraded. 5! The starting UpU point for either 3! the forward or
reaction is called the ground state, the c
 Importancia

ü  Constituyen los puntos de control de las vías metabólicas

ü  Medición de la actividad enzimática en plasma, eritrocitos o tejidos es importante

en el diagnóstico, pronóstico y tratamiento de ciertas enfermedades congénitas y
adquiridas.

ü  El aumento de la actividad enzimática es responsable de muchas enfermedades

 Importancia

ü  Mecanismo de acción de muchos fármacos

ü  Se utilizan como medicamentos. Ejemplo: L-asparaginasa

ü  Blanco de la acción de algunso venenos: cianuro

ü  Las enzimas como reactivos en determinaciones de laboratorio

ü  Deficiencia o ausencia total de una o varias enzimas en varios desordenes
genéticos hereditarios (errores congénitos del metabolismo)

ü  Herramientas en ingenieria química, tecnología de alimentos y agricultura

functional groups. (For example, a coenzyme might form a covalent intermediate
with the substrate, or an amino acid side chain might abstract a proton from the
reacting substrate.) The activated substrates and the enzyme form a transition-state
complex, an unstable high-energy complex with a strained electronic configura-

Modelo de adaptación inducida

tion that is intermediate between substrate and product. Additional bonds with the
enzyme stabilize the transition-state complex and decrease the energy required for
its formation.
The transition-state complex decomposes to products, which dissociate from the
enzyme (see Fig. 8.4D). The enzyme generally returns to its original form. The free
enzyme then binds LA CATALISIS OCURRE EN EL SITIO ACTIVO
another set of substrates and repeats the process.

A Substrate
C

Enzyme Cuando los sustratos se aproximan y

Active site Additional
bonds
se unen a una enzima, inducen un
Cofactors c a m b i o c o n f o r m a c i o n a l , u n a
Free enzyme Transition-state complex modificación análoga a colocar una
mano (sustrato) dentro de un guante
B D (enzima)
Substrate
binding site
Products

Enzyme –substrate complex Original enzyme

FIG. 8.4. Reaction in the enzyme active catalytic site. A. The enzyme contains an active
catalytic site, shown in dark red, with a region or domain where the substrate binds. The
active site also may contain cofactors, nonprotein components that assist in catalysis. B. The
substrate forms bonds with amino acid residues in the substrate-binding site. Substrate binding
induces a conformational change in the active site. C. Functional groups of amino acid resi-
dues and cofactors in the active site participate in forming the transition-state complex, which
is stabilized by additional noncovalent bonds with the enzyme, shown in red. D. Because the
Enzimas: Mecanismo de acción
Fijación de 3 puntos
58 SECCIÓN I Estructuras y funciones de proteínas y enzimas

4
LAS
PO
1 3 Los n
1

3
ben el
por ej
2 no, la
2
reord
Sitio de enzima Sustrato den p
oxida
FIGURA 7–1 Representación planar de la “fijación de tres puntos”
regula
de un sustrato al sitio activo de una enzima. Aunque los átomos 1
y 4 son idénticos, una vez que los átomos 2 y 3 se unen a sus sitios su me
complementarios en la enzima, sólo el átomo 1 puede unirse. De este rio, se
H AND TEMPERATURE
ost enzymes is plotted as a function of the pH of the reaction, an
Sitio activo
rate is usually observed as the pH goes from a very acidic level
ange; a decrease of reaction rate occurs as the pH goes from the

NAD+
R
O
+ -Alineación óptima del sustrato con los
N
C cofactores, grupos prostéticos y cadenas laterales
de los residuos de aminoácidos que realizan la
NH2
Zn2
+
transformación del sustrato en producto.
–
H O
C H Ser -Protege al sustrato contra el agua, generando un
CH3 H O ambiente cuya polaridad, acidez o alcalinidad
pueden diferir de la que hay en el citoplasma
H
N His
Liver alcohol O
dehydrogenase
CH3CH2OH CH3C H
+ +
NAD+ NADH + H+
cohol dehydrogenase (ADH) catalyzes the oxidation of ethanol (shown
de. The active site of liver ADH contains a bound zinc atom, a serine
 en nanotecnología han
olic
ual
Enzimología
conservación de
estructural molécula
primaria única de la red mediante microscopia
de dos componentes
de transmisión de carga para varias serina proteasas. Entre los implican enzima y mo
La sensibilidad limitada de las valoraciones
participan enzimáticas
de manera tra-
Resíduos catalíticos están muy conservados
residuos más conservados
dicionales
directa
figuran los que
exige el uso de un grupo grande —o conjunto— índice
en la catálisis.
cuencia, los científicos
de de eventos cata
moléculas de enzima para producir cantidades de producto me-
misma dibles. Los datos obtenidos de este modo reflejan la capacidad
CUADRO 7–1 Secuencias de aminoácidos en la vecindad de los sitios catalítico
resi- catalítica promedio de moléculas individuales. Avances recientes
tra la en nanotecnología han hecho
Enzima posible
Secuencia observar
alrededor de la—por
serina lo
S general Secuenc
la red mediante
Tripsina microscopia de fluorescencia— eventos catalíticos que
D S C Q D G S G G P V V C S G K V
re los implican enzima y molécula de sustrato individuales. En conse-
Quimotripsina A S S C M G D S G G P L V C K K N V
anera cuencia, los científicos ahora tienen la posibilidad de medir el
índice de eventos
Quimotripsina B catalíticos
S únicos
S C M y, Gen Docasiones,
S G G los P pasos
L V C Q K N V

Trombina D A C E G D S G G P F V M K S P V
ecindad Nota:
de los sitios catalíticos de varias proteasas bovinas
Las regiones mostradas son las que están a ambos lados de los residuos serilo S e histidilo H del sitio catalítico.

na S Secuencia alrededor de la histidina H

G P V V C S G K V V S A A H C Y K S G

G P L V C K K N V V T A A H G G V T T

G P 07.indd
Murray_ L V 63 C Q K N V V T A A H C G V T T

G P F V M K S P V L T A A H C L L Y P
zes the reaction S → P also catalyzes the reaction 6–3). Addition
The role of enzymes is to accelerate the inter- ten exist in th
Energía de activación
sion of S and P. The enzyme is not used up in the The interconv
s, and the equilibrium point is unaffected. How- diates thus co
he reaction reaches equilibrium much faster when steps occur in
by the step (o
Transition state (‡) ergy; this is c
case, the rate-
Reducen la energía de activación
‡ the diagram fo
Free energy, G

!Guncat
‡ the rate-limiti
‡
!Gcat
and for many
ES EP activation ene
S
rate-limiting.
P
Activation
reactions. The
at which a m
Reaction coordinate

6–3 Reaction coordinate diagram comparing enzyme-

∗ In this chapter,
 Mecanismos que emplean las enzimas para facilitar la
catalisis.
1- Catálisis por proximidad:
Para que las moléculas reaccionen, deben acercarse hasta ubicarse dentro de la
distancia formadora de enlace de otra. Cuando una enzima se une a moléculas de
sustrato en su sitio activo, crea una región de concentración local alta de sustrato.

2- Catálisis acido-base:
Los grupos funcionales ionizables de cadenas laterales aminoacilo y (cuando están
presentes) de grupos prostéticos, contribuyen a la catálisis al interactuar como ácidos o
bases.
-En la catálisis específica para ácido o específica para base participan protones (H3O+) o
iones OH– (del agua). Independiente de la concentración de otro ácido o base.

-En la catálisis por ácido general o por base general, las reacciones cuyos índices
muestran capacidad de respuesta a todos los ácidos o bases presentes.

Las enzimas de la familia de la proteasa aspártica, que incluye la enzima digestiva
pepsina, las catepsinas lisosómicas y la proteasa producida por el HIV

 Mecanismos que emplean las enzimas para facilitar la catalisis

3- Catálisis por tensión:
Las enzimas que catalizan reacciones líticas que comprenden la rotura de un enlace
covalente típicamente se unen a sus sustratos en una conformación muy desfavorable
para el enlace que sufrirá la división. La tensión resultante estira o deforma el enlace al
cual se dirige; esto lo debilita y lo hace más vulnerable a división.

4- Catálisis covalente:

Comprende la formación de un enlace covalente entre la enzima y uno o más sustratos. la
modificación química de la enzima es transitoria; en el momento en que se completa la
reacción, la enzima vuelve a su estado no modificado original.
La catálisis covalente se observa con particular frecuencia entre enzimas que catalizan
reacciones de transferencia de grupo. Los residuos sobre la enzima que participa en la
catálisis covalente por lo general son cisteína o serina y, en ocasiones, histidina.
Serina proteasa quimotripsina comprende la formación previa de un intermediario de
enzima acilo covalente.

 Mecanismos que emplean las enzimas para facilitar la catalisis

Catálisis por iones metálicos:

Aproximadamente el tercio de las enzimas conocidas necesitan iones metálicos para actuar.
El metal puede estar firmemente unido a la enzima o puede encontrarse en la solución junto
con el sustrato y puede intervenir de fierentes maneras durante la catálisis. El ión metálico
puede interaccionar con el sustrato orientándolo en la posición más adecuada para que se de
la catálisis o bien puede estabilizar intermediarios de reacción cargados. También pueden
acelerar reacciones de oxido-reducción mediante cambios reversibles de sus propios estados
de reacción.
he reaction it catalyzes. As an example, the formal sys-
ematic name of the enzyme catalyzing the reaction
ATP ! D-glucose ¡ ADP ! D-glucose 6-phosphate
Clasificación internacional de las enzimas
s ATP:glucose phosphotransferase, which indicates that
catalyzes the transfer of a phosphoryl group from ATP
o glucose. Its Enzyme Commission number (E.C. num-
er) is 2.7.1.1. The first number (2) denotes the class
ame (transferase); the second number (7), the sub-
catalyzed by an enzyme.
■ With the exception of a few catalytic RNAs, all know
enzymes are proteins. Many require nonprotein
■
coenzymes or cofactors for their catalytic function.
Enzymes are classified according to the type of
reaction they catalyze. All enzymes have formal
E.C. numbers and names, and most have trivial
names.

TABLE 6–3 International Classification of Enzymes

Class no. Class name
1 Oxidoreductases
2 Transferases
3 Hydrolases
4 Lyases
5 Isomerases
6 Ligases
Type of reaction catalyzed
Transfer of electrons (hydride ions or H atoms)
Group transfer reactions
Hydrolysis reactions (transfer of functional groups to water)
Addition of groups to double bonds, or formation of double bonds by removal of groups
Transfer of groups within molecules to yield isomeric forms
Formation of C—C, C—S, C—O, and C—N bonds by condensation reactions coupled to
cleavage of ATP or similar cofactor
Clasificación internacional de las enzimas
CLASES SUBCLASES.

1- OXIDORREDUCTASAS: Deshidrogenasas, Reductasas, Oxidasas
Hidroxilasas y Peroxidasas

2- TRANSFERASAS: Transaminasas, Transfosforilasas y Transmetilasas

3- HIDROLASAS: Lipasas, Fosfatasas, Fosfodiesterasas
Peptidasas y Glicosidasas

4- LIASAS: Descarboxilasas, Aldolasas, Deshidratasas y Desaminasas

5- ISOMERASAS: Epimerasas, Mutasas

6- LIGASAS: Sintetasas, Carboxilasas
 Oxidorreductasas
acceptor and CoA as the carrier of the succinyl group.
The energy of oxidation of !-ketoglutarate is conserved
Catalizan reacciones de oxidación-reducción
in the formation of the thioester bond of succinyl-CoA:
CoA-SH
CH2 COO # NAD" CH2 COO#
NADH
CH2 CH2 " CO2
C $-ketoglutarate C S-CoA
COO#
dehydrogenase
complex
O O
$-Ketoglutarate Succinyl-CoA

%G!& ' #33.5 kJ/mol

nitrogenous waste products. these reac
Cells contain different types of aminotransferases. is broken,

Transferasas
Many are specific for !-ketoglutarate as the amino group The electr
acceptor but differ in their specificity for the L-amino form a hig
acid. The enzymes are named for the amino group donor phate prov
Catalizan la transferencia de grupos que contienen C, N, o P ate (Fig. 1
of PLP (an
COO$ COO$ negative c
#
C O H3N C H Amino
CH2 CH2
of enzyme
(see Fig. 6
CH2 CH2 the produc
COO$ COO$ substrate c
!-Ketoglutarate L-Glutamate to the acti
phosphate
PLP The incom
amino- group from
transferase
form of an
COO$ COO$ page 678,
# and aspart
H 3N C H C O
important
R R
L -Amino acid !-Keto acid
Glutamate
 Hidrólisis
Catalizan la ruptura de enlaces por la adición de una molécula de agua
 CH2OPO23"

Liasas
lactonase
H 2O
Mg 2!

O O"
M D
C
A
HCOH
A
Via oxidativa dorecta HOCH 6-Phospho-
A gluconate
6-fosfogluconato DH HCOH
A
HCOH
A
CH2OPO23"
NADP!
2!
6-phosphogluconate Mg
dehydrogenase NADPH ! H!
CO2

CH2OH
A
CP O
A
HCOH D-Ribulose
A
HCOH 5-phosphate
A
CH2OPO23"
 Isomerasas
isomerization of glucose 6-phosphate, an aldose, to fruc-
tose 6-phosphate, a ketose:
6
CH2OPO32"
6
CH2OPO32"
5 O 1
H H O CH2OH
H Mg2!
4 1 5 2
OH H phosphohexose H HO
HO OH isomerase H OH
3 2 4 3

H OH OH H
Glucose 6-phosphate Fructose 6-phosphate

DG$% #
Catalizan la racemización de isómeros ópticos o geométricos 1.7 kJ/mol

The mechanism for this reaction involves an enediol in-

 Ligasas
!
NH3
"
OOC CH2 CH2 CH COO"
L-Glutamate

ATP
glutamine
synthetase
ADP Catalizan la formación de enlaces entre C y O, S, N, con gasto
de ATP
!
O O NH3
O
"
P O C CH2 CH2 CH COO"
O" # -Glutamyl
phosphate
!
NH4
glutamine
synthetase
Pi

!
O NH3
C CH2 CH2 CH COO"
H 2N
L-Glutamine
 Enzimas simples y conjugadas


1- SIMPLES: solo contienen resíduos de aminoácidos

Ej.: Las lisozimas.


2- CONJUGADAS: requieren un componente químico adicional

Holoenzima = Apoenzima +

-Cofactor: uno o varios metales inorgánicos
-Coenzima: molécula orgánica o metaloorgánica más compleja
-Grupo prostético: coenzima o ion metálico unido covalentememte
a la enzimas

lecular na- function, before the specific reaction catalyzed was
d, Haldane
onding in-
Iones inorgánicos que sirven como cofactor
e might be TABLE 6–1
Some Inorganic Ions That Serve as
t the heart Cofactors for Enzymes
talysis. Ions Enzymes
tury, thou-
structures Cu2! Cytochrome oxidase
Fe2! or Fe3! Cytochrome oxidase, catalase,
peroxidase
K! Pyruvate kinase
Mg2! Hexokinase, glucose 6-phosphatase,
alytic RNA
pyruvate kinase
eins. Their
heir native Mn2! Arginase, ribonucleotide reductase
red or dis- Mo Dinitrogenase
is usually Ni2! Urease
component Se Glutathione peroxidase
destroyed. Zn2! Carbonic anhydrase, alcohol
quaternary dehydrogenase, carboxypeptidases
o their cat- A and B
 Coenzimas: transportadores de grupos 6.1 An Introduction to Enzymes 185

TABLE 6–2 Some Coenzymes That Serve as Transient Carriers of Specific Atoms or Functional Groups
Coenzyme Examples of chemical groups transferred Dietary precursor in mammals
Biocytin CO2 Biotin
Coenzyme A Acyl groups Pantothenic acid and other compounds
5"-Deoxyadenosylcobalamin H atoms and alkyl groups Vitamin B12
(coenzyme B12)
Flavin adenine dinucleotide Electrons Riboflavin (vitamin B2)
Lipoate Electrons and acyl groups Not required in diet
Nicotinamide adenine dinucleotide Hydride ion (:H# ) Nicotinic acid (niacin)
Pyridoxal phosphate Amino groups Pyridoxine (vitamin B6)
Tetrahydrofolate One-carbon groups Folate
Thiamine pyrophosphate Aldehydes Thiamine (vitamin B1)

ote: The structures and modes of action of these coenzymes are described in Part II.

nown. For example, an enzyme known to act in the di- class (phosphotransferase); the third number (1),
estion of foods was named pepsin, from the Greek pep- phosphotransferase with a hydroxyl group as accepto
is, “digestion,” and lysozyme was named for its ability and the fourth number (1), D-glucose as the phosphor
o lyse (break down) bacterial cell walls. Still others group acceptor. For many enzymes, a common name
 Isozimas

Versiones de una enzima distintas desde el punto de vista físico, que catalizan la misma
reacción.

Pueden mostrar diferencias sutiles de propiedades como sensibilidad a factores reguladores
particulares o afinidad de sustrato (p. ej., hexocinasa y glucocinasa) que las adaptan a
tejidos o circunstancias específicos.

 Isozimas: Lactato deshidrogenasa
Reducido
Oxidado

OH 2H" " 2e! O

O O
CH3 CH C CH3 C C
O! O!
2H" " 2e!
Lactate lactate Pyruvate
dehydrogenase

RE 13–10Enzima tetramérica compuesta por las subunidades H y/o M

An oxidation-reduction reaction. Shown here
ation of lactate to pyruvate. In this dehydrogenation, two ele
 arritmias graves, intervenciones quirúrgicas. dete
La CK está aumentada en esfuerzos físicos, y s
Isozimas: Lactato Deshidrogenasa
inyecciones intramusculares, heridas musculares y
daño hipóxico de los músculos esqueléticos. Este
nor
dete
aumento se debe casi exclusivamente a lesiones de la faci
musculatura esquelética. se d
Enzima tetramérica compuesta por las subunidades H y/o M que
Láctico Deshidrogenasa exc
ano
Tipo Composición Localización
acti
Miocardio, eritrocito,
LDH1 HHHH ha
riñón, páncreas
mio
Miocardio, eritrocito,
LDH2 HHHM trop
riñón
enz
Páncreas, pulmón,
LDH3 HHMM de e
leucocitos
la e
Hígado, músculo
LDH4 HMMM pro
esquelético
dife
Hígado, músculo
LDH5 MMMM enz
esquelético
valo
H que es posible separar co como herramientas para determinar la concentración de me-
ndo una valoración aco- tabolitos críticos; por ejemplo, la glucosa oxidasa suele utilizarse
la LDH ha quedado su- para medir la concentración plasmática de glucosa. Cada vez
por otras proteínas que
ma. Lactato Deshidrogenasa
con mayor frecuencia se emplean enzimas como recursos para
el tratamiento de lesión y enfermedad. El activador del plasmi-

+ –

o
S (Piruvato)
nasa
Corazón A

NADH + H+

Normal B

PMS oxidado

Hígado C
NBT reducido
(azul formazán)

5 4 3 2 1

ológico de isozimas de lactato deshidrogenasa (LDH) en el suero humano. Las isozimas de LDH en el
 transaminases
Enzyme reactions are multistep in nature and comprise
3. Hydrolases A-B + H2O→A-H + Alkaline phosphatase, several partial reactions
B-OH trypsin

Isozimas: Lactato Deshidrogenasa

In 1913, long before the structure of proteins was known,
4. Lyases X-A-B-Y→A = B + XY fumarase,
Leonor Michaelis and Maud Leonora Menten developed a
(synthases) dehydratases
simple model for the kinetics of enzyme-catalyzed reactions
5. Isomerases A ⇌ isoA Triose phosphate (Fig. 6.4). The Michaelis–Menten model assumes that the
isomerase,
substrate S binds to the enzyme E, forming an essential inter-
phosphogluco-mutase
mediate, the enzyme-substrate complex (ES), which then
6. Ligases A + B + ATP→A-B + Pyruvate carboxylase, undergoes reaction on the enzyme surface and decomposes to
(synthetases) ADP + Pi DNA ligases
E + P (product). The model assumes that E, S, and ES are all in

Electrophoretic analysis of
LDH isozymes on gel

A B C A Normal serum B Acute myocardial infarction C Acute hepatitis

Normal Acute Acute
serum myocardial hepatitis
+ infarction LDH2
LDH1
LDH1
LDH2 LDH3

LDH3 LDH4
LDH4 LDH5

LDH5
_ _ + _ + _ +
Fig. 6.3 Densitometric patterns of the lactate dehydrogenase (LDH) isozymes in serum of patients diagnosed with myocardial infarc-
tion or acute hepatitis. Isozymes, differing slightly in charge, are separated by electrophoresis on cellulose acetate, visualized using a chromogenic
substrate, and quantified by densitometry. Total serum LDH activity is also increased in these patients. Since hemolysis releases LDH from red blood
cells and affects isozyme distribution and differential diagnosis, blood samples should be treated with care. The LDH measurements for the diagnosis
of myocardial infarction have now been superseded by assay of plasma troponin and other biomarkers.
 Isozimas: Creatin cinasa

Enzima dimérica compuesta por dos subunidades B y o M

Tipo o Nombre Composición Tejido

CK1 BB Cerebral

CK2 MB Cardíaca

CK3 MM Muscular
Enzimas y proteínas en infarto de miocardio

Corazón das de maner

teria pueden
Arteria coronaria factores de cr
ej., factor de
Trombo acumulan pr
no y calcio e
de LDL oxid
Infarto ticular impor
crófagos (cél
Liberación de
troponinas, CK-MB,
de crecimien
Mb, AST, ALT, LDH-1 en la ateroscl
y otras proteínas y crófagos y lin
enzimas, hacia la na C reactiva
circulación
crónica. A m
tría adiposa e
FIGURA 57–19 Diagrama de un trombo en una arteria ber inflamaci
 Myoglobin (FD
Myoglobin is a heme-containing protein that binds oxygen within cardiac my
and skeletal muscle; only a single form is common to both muscle types. 20
Enzimas y proteínas en infarto de miocardio
Having a molecular weight of only 18 kDa, myoglobin apparently leaks affi
from damaged cells more rapidly than other proteins. Following MI, a s
ari
em
Myoglobin, or bef
CK-MB isoform ratio to
cle
Transaminase, total CK,
sen
or CK-MB tha
100
Marker concentration

LD-1 con
(% of maximum)

80 on
at
60 cTnI pre
est
40 or cTnT
20 AS
0 Th
0 24 48 72 96 120 144
tot
Hours after onset of chest pain sep
cT
Figure 18-1 Schematic depiction of the kinetics of several cardiac markers fol-
 gicos, posible consumo de fármacos o drogas, así como la sensi-
bilidad y la especificidad diagnóstica de la prueba enzimática.
NZIMAS
Priincipales enzimas en clínica
CUADRO 7–2 Principales enzimas séricas usadas
en el diagnóstico clínico
ha desempeñado Enzima sérica Principal uso diagnóstico
varios procesos
Aminotransferasas
funcionales de la
linesterasa, lipo- Aspartato aminotransferasa Infarto de miocardio
(AST o SGOT)
Alanina aminotransferasa (ALT Hepatitis viral
o SGPT)
+ Amilasa Pancreatitis aguda

Ceruloplasmina Degeneración hepatolenticular

+
(enfermedad de Wilson)

Creatina cinasa Trastornos musculares e infarto

de miocardio

γ-Glutamil transferasa Diversas enfermedades hepáticas

Isozima 5 de la lactato Enfermedades hepáticas
+ H+
deshidrogenasa

Lipasa Pancreatitis aguda

a para la actividad
mediante la Fosfatasa ácida Carcinoma metastásico
de la próstata
cto por la glucosa-
adida y NADP+. Fosfatasa alcalina (isozimas) Diversos trastornos óseos,
genasa, el índice enfermedades hepáticas obstructivas
m, está regido por
 Enzimas: cinética
186 Enzymes
FIGURE 6–1 Binding of a substrate to an enzyme at the active site.
The enzyme chymotrypsin, with bound substrate in red (PDB ID
7GCH). Some key active-site amino acid residues appear as a red
where E, S, and P represent the enzyme, s
product; ES and EP are transient complex
zyme with the substrate and with the prod
To understand catalysis, we must firs
Noé Rigoberto Rivera. MD
the important distinction between reacti
Biología Molecular Ph.D
and reaction rates. The function of a cata
crease the rate of a reaction. Catalysts d
Departamento de Bioquímica
reaction equilibria. Any reaction, such as
Facultad de Medicina
described by a reaction coordinate diagram
pictureUniversidad de El Salvador
of the energy changes during the rea
cussed in Chapter 1, energy in biological s
scribed in terms of free energy, G. In th
diagram, the free energy of the system is pl
the progress of the reaction (the reaction
The starting point for either the forward o
reaction is called the ground state, the
to the free energy of the system by an aver
(S or P) under a given set of conditions.
 ACTIVIDAD ENZIMATICA

1- NUMERO DE RECAMBIO
Es el número de moléculas de sustrato transformadas por unidad de tiempo
por una molécula de enzima.

2-UNIDAD ENZIMATICA (UI)

La cantidad de enzima que es capaz de catalizar la transformación de 1
micromol de sustrato o coenzima por minuto por litro de suero en condiciones
estándar (tiempo, temperatura, pH, concentración de sustrato, presencia de
activadores y concentración iónica.

3- KATAL

La cantidad de enzima que es capaz de catalizar la transformación de 1 mol de

sustrato por segundo.

4- LA ACTIVIDAD ESPECÍFICA es el número de unidades de enzima por miligramo

de proteína (U/mg prot) o por mililitro de disolución (U/ml). da una idea de la
pureza de la enzima.
 Como medir la actividad enzimática

-Medir el aparecimiento de un producto

-Medir a desaparición de un sustrato

-Medir cambios en el estado de oxidación de una coenzima
Factores que afectan la actividad enzimática

Temperatura:
-Las enzimas necesitan estabilidad
-psicrófilos Salinidad
para enfrentar las condiciones en
-mesófilos -osmofilos las que se desarrollan
-termófilo
-Organismo al que pertenece

-Ambiente de trabajo
pH
Presion -acidófilos En sus condiciones naturales las
-barófilos -neutrófilos e n z i m a s p r e s e n t a n a c t i v i d a d ,
-alcalófilos e s p e c i f i c i d a d d e s u s t r a t o ,
enantioselectividad y estabilidad
características
 Temperatura

-Psicrófilos
Moderados Extremos Hipertermófilos
-Mesófilos
≥ 45⁰C ≥ 65⁰C ≥ 80⁰C
-Termófilo
 Eecto de la Temperatura
Enzymatic Reactions 47

Temperatura óptima

Es necesario respetar las

t e m p e r a t u r a s q u e v i e n e n
indicadas en los protocolos de
laboratorio

Se mide la actividad de la enzima a diferentes temperaturas

10 20 30 40 50
Temperature (°C)

Figure 4.11 Temperature dependence of a typical

enzymatic reaction. V, Reaction rate.
PCR en Baño de María

u  ADN polimerasas de mesófilos

u  Agregar ADN pol en cada ciclo

u  Cambiar los tubos a cada temperatura

u  Desnaturalización

u  Hibridación primers

u  Extensión (síntesis DNA)
Kary Mullis Premio Nobel 1993

1985: PCR
Polimerasas Termoestables
ADN polimerasas termoestables:
• activas después de ciclos a 95ºC
• Temperatura óptimo a 72ºC

u  ADN polimerasas muy procesivas: 1

minuto por kb

Thermus aquaticus (Taq pol) y derivados

Thermus thermophilus (Tth pol)

u  ADN polimerasas capaces de realizar
corrección de errores
1.5 minutos por kb
Pyrococcus furiosus (Pfu)

Thermococcus litoralis (Vent)

Termociclador para PCR a Punto Final

u  Calentar la tapa

u  Desnaturalización

u  Hibridación primers

u  Extensión (síntesis DNA)

 ns Competitive inhibitors are structurally related to the nor-
ve mal substrate of the enzyme. They compete with the sub-
m-
es,
pH óptimo
strate by binding noncovalently to the active site of the
his

ns V
Alkaline
er. Pepsin phosphatase
ns
Lysozyme
to
r-
r-
a
a-

r- 2 4 6 8 10
pH
to
ks Figure 4.12 pH dependence of some enzymes. V, Reaction
ot rate.
Se mide la actividad de la enzima a diferente pH
Efecto de la concentración de sustrato
Fórmula de Michaelis-Menten
 Concentración de sustrato
CAPÍTULO 8 Enzimas: cinética
Km es la concentración de sustrato a la cual la Vi es la mitad de la Vmax alcanzable a una
concentración particular de enzima

máx
Saturada
V. Máxima
máx
Hipérbola
v
Velocidad de la Rx

máx

V Inicial

Km y Vmax son concentraciones de sustrato

FIGURA 8–4 Efecto de la concentración de sustrato sobre
 Concentración de sustrato
CAPÍTULO 8 Enzimas: cinética
Km en algunos casos puede considerarse una medida de la afinidad de una enzima por el
sustrato

máx
Saturada

máx

v Km: mitad de la Vmax

máx

V Inicial

FIGURA 8–4 Efecto de la concentración de sustrato sobre

La cinética de Michaelis-Menten la cumplen las enzimas no reguladoras
ustratos, los principios que se comentan a con- ES. Dado que no queda enzima libre disponible
ican con igual validez. Más aún, al emplear condi- los aumentos adicionales de [S] no pueden aum
primer orden (véase antes), los científicos pueden de la reacción. En estas condiciones de saturació

Concentración de sustrato
endencia del índice de reacción en un reactivo
medio de la selección apropiada de sustratos fijos
sólo de —y, de este modo, está limitada por— la
cual el producto se disocia de la enzima de modo
otras palabras, en condiciones de seudoprimer binarse con más sustrato.

=S

=E

A B C

FIGURA 8–5 Representación de una enzima en la presencia de una concentración de sustrato que
está por debajo de Km (A), a una concentración igual a Km (B), y a una concentración bastante por arriba
Cinética de primer orden Cinética de orden cero
de K (C). Los puntos A, B y C corresponden a esos puntos en la figura 8-4.
m
 Th
Concentración de sustrato maxim
can b
strate
Le
Zero-order kinetics Vmax,
(rate does not depend bindi
Initial velocity (V init)

on concentration forms
of substrate)

First-order kinetics
(rate depends on
concentration of
substrate) wher
Substrate concentration [S]
D[ES
the co
nattainable because of solubility limits or the cost of the substrate. The ex-
olated line can be used to obtain Vmax.
Gráfica de dobles recíprocos
1 KM 1 1
V
=
V max
( [S]
(+ V max
Ecuación de Lineweaver-Burk

1
V

KM
Slope =
–1 V max
x intercept =
KM
1
y intercept = ■ FIGURE 6
V max reciprocal
of reaction
reciprocal
0 1
[S]. The sl
[S] intercept is
Límea recta para las enzimas que cumplen la cinética de Michaelis-Menten
diluir la solución madre por factores de 1:2, 1:3,
s datos entonces caerán en el eje 1/[S] a intervalos
Gráfico del doble recíproco o de lineweaver-Burk
, etc. De manera alternativa, para
minimizar la

Km
1 Pendiente =
vi Vmáx -Permite determinar Vmax con más
precisión


– 1
Km 1 -La facilidad con la cual puede usarse
Vmáx para determinar los mecanismos
cinéticos de inhibidores enzimáticos
0 1
[S]

Gráfico del doble recíproco o de Lineweaver-Burk

aposición con 1/[S] usado para evaluar la Km y Vmáx.
 Km
TABLE 6–6 Km for Some Enzymes and Substrates
Enzyme Substrate Km (mM)
Hexokinase (brain) ATP 0.4
D-Glucose 0.05
D-Fructose 1.5
Carbonic anhydrase HCO%
3 26
Chymotrypsin Glycyltyrosinylglycine 108
N-Benzoyltyrosinamide 2.5
"-Galactosidase D-Lactose 4.0
Threonine dehydratase L-Threonine 5.0
 k2 "" k!1, has units of reciprocal time. It is also called the
!1 are com- turnover number. It is equivalent to the number of
nction of all
elis-Menten
K
substrate molecules converted to product in a given unit
cat molecule when the enzyme is
of time on a single enzyme
avior of the saturated with substrate. The turnover numbers of sev-
Es el número de moléculas de sustrato transformadas por unidad de tiempo por
ed a simple eral enzymes are given in Table 6–7.
una molécula de enzima.

TABLE 6–7 Turnover Numbers, kcat, of Some Enzymes

Enzyme Substrate kcat (s!1 )
Catalase H2O2 40,000,000
Carbonic anhydrase HCO%
3 400,000
Acetylcholinesterase Acetylcholine 14,000
"-Lactamase Benzylpenicillin 2,000
Fumarase Fumarate 800
RecA protein (an ATPase) ATP 0.5
equation and the constant kcat /Km is a second-order rate Km ! 40 mM $ 50 mM
constant with units of M#1s#1. There is an upper limit to
Km $ 50 mM # 40 mM
kcat /Km, imposed by the rate at which E and S can dif-

Constate de especificidad
fuse together in an aqueous solution. This diffusion-
controlled limit is 108 to 109 M#1s#1, and many enzymes
have a kcat /Km near this range (Table 6–8). Such enzymes
are said to have achieved catalytic perfection. Note that
different values of kcat and Km can produce the
maximum ratio.
Km $ 10 mM
Once you have worked with this equation, you will
recognize shortcuts to solve problems like this. For
example, one can calculate Vmax knowing that kcat[Et] $
Vmax (600 s#1 % 0.020 !M $ 12 !M s#1 in this case). A

TABLE 6–8 Enzymes for Which kcat/Km Is Close to the Diffusion-Controlled Limit (10 8 to 10 9 M#1s#1)
kcat Km kcat /Km
Enzyme Substrate (s!1 ) (M) (M!1 s!1 )
Acetylcholinesterase Acetylcholine 1.4 % 104 9 % 10#5 1.6 % 108
Carbonic anhydrase CO2 1 % 106 1.2 % 10#2 8.3 % 107
HCO#3 4 % 105 2.6 % 10#2 1.5 % 107
Catalase H2O2 4 % 107 1.1 % 100 4 % 107
Crotonase Crotonyl-CoA 5.7 % 103 2 % 10#5 2.8 % 108
Fumarase Fumarate 8 % 102 5 % 10#6 1.6 % 108
Malate 9 % 102 2.5 % 10#5 3.6 % 107
"-Lactamase Benzylpenicillin 2.0 % 103 2 % 10#5 1 % 108

Source: Fersht, A. (1999) Structure and Mechanism in Protein Science, p. 166, W. H. Freeman and Company, New York.
 La ecuación de Hill: Enzimas que muestran
unión cooperativa de sustrato
SECCIÓN I

Estructuras y funciones de proteínas y enzimas

∞ de sustrato llamada S50, l

Curva sigmoidea resultado la mitad de la v
análoga a la P50 para la un
Propia de enzimas multiméricas que unen
sustratos en múltiples sitios

vi EL ANÁLISIS C
DISTINGUE EN
INHIBICIÓN CO
Y NO COMPET
Los inhibidores de las act
0 ∞
cionan tanto agentes farm
[S]
gación para estudiar el m
A 8–7 Representación de cinética de saturación fuerza de la interacción e
 finidad de los sitios restantes para unión a sulfato unión a sustrato
La ecuación de Hill: Enzimas que muestran
l. Mientras mayor es el valor para n, más alto es el grado sustrato. Por en
eración, y más sigmoideo será el gráfico de vi contra [S]. competitivos cl
unión cooperativa de sustrato
pendicular trazada desde el punto donde el término y un sustrato y, as
máx
− vi) es cero interseca el eje x en una concentración de la enzima su
inhibición com
la pendiente de la línea n es el coeficiente de Hill:
deshidrogenasa
geno de cada u
n: 1 todos los sitios de unión son independientes
1 (figura 8-9). T
n: > 1, se dice que la enzima
vi

muestra cooperación positiva malonato (−OO

vo  de la succin
Vmáx –
vi

0 Pendiente = n
ES o uno EI, re
nato sólo conti
Log

–1 S50: la concentración de sustrato que da por

resultado la mitad de la velocidad máxima
H

–4 S50 –3 H C

Log [S] –
OOC C
 Inhibición enzimática reversible
-Inhibidor competitivo:
Se une al sitio activo, la mayoria son análogos de sustrato, un inhibidor competitivo
aumenta K'm y no tiene efecto sobre Vmáx

-Inhibidor no competitivo:
la unión del inhibidor no afecta la unión de sustrato, disminuyen Vmax pero no afectan Km


-Inhibidor acompetitivo:
Se fija a un sitio distinto del que se fija el sustrato al sitio acttivo ya que solo se une al
complejo ES


-Inhibidor mixto:
Se fija a un sitio distinto al sustrato, pero se fija tanto a E como a ES
 V Vmax KI 3S 4 Vmax
y5 m 3 x 1 b (6.18)

Inhibición competitiva
Here the term 1/V takes the place of the y coordinate, and the term 1/[S] takes
the place of the x coordinate, as was the case in Equation 6.17. The intercept
1/Vmax, the b term in the equation for a straight line, has not changed from the

Un inhibidor competitivo actúa al disminuir el número de moléculas de enzima libres

disponibles para unión a sustrato

+2[I]
1 +[I] KS
V No
inhibitor E ES
–1 (–I)

KM (1 + [I]
K
( I
–1
KM 1
KI
Vmax
E EI
0 1
[S]

No tiene efecto sobre V máx pero aumenta K'm

■ FIGURE 6.12 A Lineweaver–Burk double-reciprocal plot of enzyme kinetics
for competitive inhibition.

Estatinas que actúan como inhibidores competitivos de la HMG-CoA reductasa

. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s).

 V Vmax 3S 4 Vmax
y5 m 3 x 1 b

Inhibición no competitiva
In noncompetitive inhibition, we replace the term Vmax with the expression for
I
Vmax , to obtain
1 KM 3I 4 1 1 3I 4
5 a11 b 3 1 a11 b (6.19)
V Vmax KI 3S 4 Vmax KI
y5 m 3 x 1 b
Noncompetitive inhibition

+I
KM
KI 1 Slope =
Vmax
(1 + [I]
K I
(
V
E EI –I
1 [I]
KS KS
Vmax
( 1 +
KI
( KM
Slope =
Vmax

K! I 1 1
–
KM Vmax
ES ESI

0 1
[S]

■ FIGURE 6.13 A Lineweaver–Burk plot of enzyme kinetics for noncompetitive inhibition.

 mmon
n. The KI
tained
riation
rature
EI
Inhibidor acompetitivo I I

a reac- (b) Uncompetitive inhibition

ctions,
r a ter- E!S ES E!P
6–14). !
more I
scope S
ce one KI" S
udying
ESI
d reac- I
ividual
nzyme I
ng the S
y reac-
ts dur- (c) Mixed inhibition
served.
y short, E!S ES E!P
d reac- I
ividual
nzyme
ng the Inhibición mixta I
S
y reac-
ts dur- (c) Mixed inhibition
erved.
short, E!S ES E!P
ues for ! !
gain a I I
hanges S
action KI KI" S
hanges
he rate EI ! S ESI
estiga- I I
ividual
ples of S
ded in S
I I

FIGURE 6–15 Three types of reversible inhibition. (a) Competitive in-

hibitors bind to the enzyme’s active site; KI is the equilibrium constant
Inhibición enzimática reversible

Inhibidor Vmax Km

Competitivo Normal Alta

No competitivo Afectada Normal

Acompetitivo Alterada Alterada

Mixto Alterada Alterada

 Inhibición irreversible

u  Forman o rompen enlaces covalentes con residuos aminoacilo esenciales para la unión a
sustrato, catálisis o mantenimiento de la conformación funcional de la enzima.

u  La enzima permanece inhibida incluso después de eliminar el inhibidor del medio

circundante


u  Caso especial: ¨inactivadores suicidas o inactivadores basados en el mecanismo, utilizan
el mecanismo de reacción enzimático normal para inactivar la enzima.
 H C R groups
C N CH3 Penicillin V
O CH

b-Lactam COOH

Penicilinas
ring
HO CH

NH2

Amoxicillin

General structure of penicillins

(a)
Inhibidores irreversibles de las transpeptidasas que participan en la sintesis de
peptidoglicanos de la pared bacteriana
6.4 Examples of Enzymatic Reactions 217
O H H
S CH3
R C N C C
H C
C N
CH2 O CH CH3
Trans-
peptidase Ser OH COOH Penicillin
Penicillin G
Side chain Thiazolidine ring (benzylpenicillin)

O H H
S CH3 O CH2
R C N#C C! O H H
!

H C R groups
C N CH3 Penicillin V S CH3
O CH R C N C C
H C
b-Lactam COOH Trans- C N
CH CH3
ring peptidase Ser O H
HO CH O
COOH
NH2
Stably derivatized,
Amoxicillin inactive transpeptidase

General structure of penicillins

(b)

(a)
FIGURE 6–27 Transpeptidase inhibition by !-lactam antibiotics. (a) by injection. Penicillin V is nearly a
!-Lactam antibiotics feature a five-membered thiazolidine ring fused can be administered orally. Amoxi
to a four-membered !-lactam ring. The latter ring is strained and in- tiveness, is readily administered or
218
β-Lactamasas
Enzymes

O H
S CH3 H2C
R C N C C
H C
C N C
O CH CH3 O
b-Lactamase Ser OH COOH Penicillin

O H H
S CH3
R C N C C
H C b-Lactamase Ser OH
C N
b-Lactamase Ser O H CH CH3
O
COOH
H2O

b-Lactamase

O H H b-Lactamase Ser O
S CH3
R C N C C
H C O
C N
!
O H CH CH3
O
COOH
Inactive penicillin Nu
(a)
 Inhibidores de β-Lactamasas
H
CH3 O CH2OH
H2C C
C C
C N H
CH3 O CH

H Penicillin COOH Clavulanic

acid

H Ácido clavulánico
O CH2OH
CH3 H2C C
b-Lactamase Ser OH C C
C C N
CH H
H CH3 O "
COOH H
OOH

actamase
H HH
O CH2OH
b-Lactamase Ser O C C
C CH
CH3 C N
CH H
C O "
H COOH
H CH3
OOH
llin Nu
H H
O CH2OH
b-Lactamase Ser O C C
inhibition. (a) !- C CH
C HN H
g in !-lactam antibi- CH
O
a suicide inhibitor, COOH
!-lactamases to cre-
ve species is attacked
the enzyme.
Inactive b-lactamase
nactivates the !-
(b)
 Proteasa del VIH
Es una aspartil proteasa: dos resíduos de aspartato del sitio activo facilitan el ataque
directo del agua sobre el enlace peptídico a hidrolizar
6.4 Examples of Enzymatic Reactions 219

Aided by general The tetrahedral

base catalysis, water intermediate O
C Peptides
O attacks the carbonyl O collapses; the
C carbon, generating C amino acid leaving C
a tetrahedral group is protonated
C intermediate. C as it is expelled. HN
C N C N C OH
C C C
!
O O OH O
O OH O O O H O OH
C H H O! C HO C O!
C C C
Asp2 5 Asp2 5 Asp2 5 Asp2 5 Asp2 5 Asp2 5
O O O
HIV protease

FIGURE 6–29 Mechanism of action of HIV protease. Two active-site facilitating the attack of water on the peptide bond. The unstable tetra-
Asp residues (from different subunits) act as general acid-base catalysts, hedral intermediate in the reaction pathway is highlighted in pink.
Especialmente enlace peptídico entre fenilalanina y prolina
 C
metalloproteases doleaving
not. The HIV protease is an
amino acid C as- HO
artyl group is protonated
C protease. Two active-site Asp residues facilitate a
as it is expelled. HN Indinavir
irect attack of water on the peptide bond to be cleaved
N OH
Fig. 6–29). The initial product ofC the C attack of water

ave seen
Inhibidores de proteasas del VIH
n the carbonyl group of the peptide bond to be cleaved
OH
an unstable tetrahedral O intermediate,
OH
O
much as we S O
H
NC(CH3 )3
HO for the chymotrypsin C reaction. This interme-
O! CH3 O
•CH3 SO2 — OH
iate is close C in structure and energy to the reaction
C HO
25 Asp2 5 N N
ansitionO state.
Asp The drugs that have been developed
O as 2 5
Asp H
Forman complejos no covalentes con la enzima, pero se unen tan fuertemente que son
IV protease inhibitors form noncovalent complexes OH
ithfacilitating
the enzyme, butofthey
water bind
on theto
considerados inhibidores irreversibles
the attack it sobond.
peptide tightly
The that they
unstable tetra-
an hedral
be considered irreversible inhibitors. The tight
intermediate in the reaction pathway is highlighted in pink. Nelfinavir
inding is derived in part from their design as transi-
on-state analogs (see Box 6–3). The success of these
rugs makes a point worth emphasizing. The catalytic
rinciples we have studied in OHthis chapter are not sim- OH
N CH3 O
ly abstruse ideas to be memorized—their H application H
N N O N N NH
aves lives.N N
H
The HIV protease Hcleaves peptide O bonds between •H2 SO4 O O
NC(CH3 )3 CH 3
he and Pro residues O most efficiently. The active site H3 C CH3
hus has a pocket to bind aromatic groups HO next to the
Indinavir Lopinavir
ond to be cleaved. The structures of several HIV protease
nhibitors are shown in Figure 6–30. Although the struc-
ures appear varied, they all share a core structure—a
main chain with aS hydroxyl group Hpositioned next to a
O NC(CH3 )3 O
ranch containing
CH3 O a benzyl group. This arrangement
•CH3 SO2 — OH
tar- H
N N
etsHOthe benzyl group to the aromatic binding pocket. The N N
H
djacent hydroxyl N Hgroup N
mimics the negatively charged O NH2
OH H
xygen in the tetrahedralOH intermediate in the normal reac-
O NC(CH3 )3
on, providing a transition-state analog. The remainder of O
Saquinavir
achNelfinavir
inhibitor structure was designed to fit into and bind
o various crevices along the surface of the enzyme, en- FIGURE 6–30 HIV protease inhibitors. The hydroxyl group (red) acts as
 ENZIMAS: Regulación
186 Enzymes
FIGURE 6–1 Binding of a substrate to an enzyme at the active site.
The enzyme chymotrypsin, with bound substrate in red (PDB ID
7GCH). Some key active-site amino acid residues appear as a red
where E, S, and P represent the enzyme, s
product; ES and EP are transient complex
zyme with the substrate and with the prod
To understand catalysis, we must firs
Noé Rigoberto Rivera. MD
the important distinction between reacti
Biología Molecular Ph.D
and reaction rates. The function of a cata
crease the rate of a reaction. Catalysts d
Departamento de Bioquímica
reaction equilibria. Any reaction, such as
Facultad de Medicina
described by a reaction coordinate diagram
pictureUniversidad de El Salvador
of the energy changes during the rea
cussed in Chapter 1, energy in biological s
scribed in terms of free energy, G. In th
diagram, the free energy of the system is pl
the progress of the reaction (the reaction
The starting point for either the forward o
reaction is called the ground state, the
to the free energy of the system by an aver
(S or P) under a given set of conditions.
 Enzimas reguladoras
-Las enzimas reguladoras exiben un aumento o disminución de la actividad catalítca en
respuesta a ciertas señales

-Enzimas alostéricas: responden a la unión reversible no covalente de moduladores
alotéricos positivos o negativos

-Enzimas reguladas por modificación covalente reversible

-Cada vía posee almenos dos enzimas reguladoras

-Suelen ser proteínas con múltiples subunidades, el sitio regulador y el sitio activo se
encuentran en subunidades separadas.

-algunas enzimas estan moduladas por proteinas reguladoras

-otras se activan por proteólisis selectiva irreversible
Mecanismos de regulación enzimática
Tipo de modulación

Alostética

Covalente reversible

Rotura proteólitica

Union a proteínas reguladoras

Síntesis de nuevas moléculas

Degradación enzimática

Compartamentalización subcelular
572
Regulación enzimática
Principles of Metabolic Regulation

1 Extracellular
signal

Receptor Enzyme undergoes

9 phosphorylation/dephosphorylation
Transcription Enzyme combines
factor 10 P
with regulatory protein

L
L
Transcription of Enzyme binds
2
specific gene(s) kinase phosphatase ligand L
8
mRNA (allosteric
L effector)
Regulatory
DNA protein S
Nucleus Enzyme S
3 mRNA degradation
Product Enzyme binds
7
substrate S

Enzyme sequestered
6
in subcellular organelle

Endoplasmic reticulum

Protein degradation
5
4 mRNA translation on ribosome (ubiquitin; proteasome)
 Regulación alostérca
idine nucleotides (
S Substrate
chains organized in
M Positive modulator
C R Figure 6–32 show
Less-active enzyme
enzyme, deduced f

-Enzimas homotrópicas: si el modulador es el

"M !M
mismo sustrato In Many Pathways, R
Allosteric Enzymes
-Enzimas heterotropicas: si el modulador es
S C R M diferente al sustrato In some multienzym
More-active enzyme specifically inhibited
whenever the conce
the cell’s requireme
action is slowed, sub
ferent rates as their
S C R M Active
enzyme-substrate of production of th
complex brought into balanc
 K0.5
[S] (mM)
Regulación alostérca
(a)

Vmax

Modulador +
V0 ( ! M/min)

1 Modulador -
" 2 Vmax

K0.5
#
K0.5 K0.5
"

[S] (mM)
 Enzymes
Regulación alostérca
(a) interfac
Vmax
larly w
High-activity hemogl
R state
Modulador + the con
actions
V0 (m /min)

AT
1 Low-activity Modulador - heterot
2 Vmax T state the sub
the enz
relative
This ac
K0.5 change
[S] (m )
sigmoid
 this sequence inhibits threonine dehydratase, nor is any
other enzyme in the sequence inhibited by isoleucine.

Inhibición por retroalimentación

Isoleucine binds not to the active site but to another spe-
cific site on the enzyme molecule, the regulatory site.

COO"
! A
H3NO CO H
A L-Threonine
HO CO OH
A
CH3

threonine
E1
dehydratase

A El producto final de la vía inhibe a la primera enzima

E2

E3 FIGURE 6–33 Feedback inhibi-

tion. The conversion of L-threo-
C nine to L-isoleucine is catalyzed
by a sequence of five enzymes (E1
E4
to E5). Threonine dehydratase (E1)
ranscar- D is specifically inhibited allosteri-
atory en- cally by L-isoleucine, the end
catalytic E5 product of the sequence, but not
gulatory by any of the four intermediates
and yel- COO" (A to D). Feedback inhibition is
! A
ounding H3NO CO H indicated by the dashed feedback
A
re on the line and the ! symbol at the
HO CO CH3 L-Isoleucine
es in en- A threonine dehydratase reaction
ucleotide CH2 arrow, a device used throughout
A
er 22. CH3 this book.
 Modificación covalente reversible

Existen más de 500 tipos de modificación covalente reversible.

Fosforilo, acetilo, metilo, amida, carboilo, miristilo, palmitilo, prenilo, hidroxilo, sulfato,
ribosilo, ubiquitina y sumo.
 n out of the active site, alter the enzyme’s in-
ith other proteins, or force conformational
Modificación covalente reversible
at translate into changes in Vmax or Km. For

Protein
substrate
-La fosforilación es probablemente el tipo de
Ser/Thr/Tyr OH modififcación covalente más importante
Histidina
-Un tercio de todas las proteínas de eucariotes
ATP Pi sufre fosforilación

phosphoprotein
protein kinase -la fosforilación de un resíduo afecta la
phosphatase
estructura y actividad catalítica de las enzimas
ADP H2O
O
Ser/Thr/Tyr O P O–
O–
 Exposición de Tirosinas
-la fosforilación de un resíduo afecta la estructura y actividad catalítica de las enzimas
 has persisted in common usage and in the literature.) glucose 1-phos
The enzyme (phosphorylase b kinase) responsi- are adequate,
ble for activating phosphorylase by transferring a phos-
Activación de la glucógeno fosforilasa
AMP binds, ina
phoryl group to its Ser residue is itself activated by When the
phosphorylas
protein phosp
Ser14 OH OH Ser14
side side ryl groups from
chain CH2 CH2 chain less active form
Like the e
Phosphorylase b
(less active) phorylase of l
Menos activa phorylation/de
dephosphoryla
glucagon
(liver)
blood glucose
2Pi 2ATP the cascade m
phosphorylase a phosphorylase b
phosphatase kinase
(PP1)
2H2O 2ADP epinephrine, FIGURE 15–34 Reg
[Ca2+], [AMP]
(muscle) lent modification.
lase a, Ser14 resi
P P
Phosphorylase a is
O O
by enzymatic loss
CH2 CH2
rylase a phosphata
Phosphorylase a PP1). Phosphoryla
(active) Activa lase a by the actio
glycogen phospho
raction of an SH2 domain with a P –Tyr residue in
PDB ID 1SHC) The SH2 domain is represented as a
232
Múltiple osforilación de la Glucógeno sintasa
Enzymes
r. The phosphorus of the phosphate group in the in-
visible as an orange sphere; most of the Tyr residue
iew. The next few residues toward the carboxyl end
in are shown in red. The SH2 domain typically 3 in-
Phosphorylation
yr (which is assigned the index position 0) and the
sites on 2 (designatedA !1,B !2,
C 4
toward the carboxyl terminus
glycogen
mains (Src, Fyn, Hck,!Nck) favor negatively charged
synthase H 3N
and !2 positions; others (PLC-!1, SHP-2) have a
roove that binds to aliphatic residues in positions
ifferences define subclasses of SH2 domains that
larly, glycogen synthase kinase 3 (GSK3) is inactive
when phosphorylated on a Ser residue in its autoin-
hibitory domain (Fig. 12–22b). Dephosphorylation of
that domain frees the enzyme to bind (and then phos-
phorylate) its target proteins.
1 inhibits trypsin. !1-Antiproteinase (Mr 5
5 regiones, al menos 9 de fosorilación
5(a) A B
inhibits neutrophil
(b) elastase (neutrophi
SH3 Pro
leukocyte, or white blood cell; elastase i
COO "
ing on elastin, a component of some
Ser OH
sues). An insufficiency of !1-antiprotei
SH2
Degree ofP Tyr be caused by exposure to cigarette sm

Inactivada
er specificities. Phosphorylation synthase associated with lung damage, including
Kinase sites inactivation Proteases are not the only protei
HO –Tyr Active; substrate Ser P
Active proteolysis.
positioned for In other cases, however, th
Active
Protein
is bound inkinase
a deep A pocket in
1A,an1B,
SH22,do-
4 site ! HO –Tyr
called not zymogens
phosphorylation Ser OHbut,site
more general
h of its phosphate
Protein kinase Goxygens participating
1A, 1B, 2 ! HO –Tyr
Src
or proenzymes, SerasOH
appropriate.
GSK3
For ex
nding or electrostatic interactions; the
Protein kinase C 1A ! nective tissue protein collagen is initiall
s on two Arg residues figure prominently
Ca2!differences
Subtle /calmodulin in the structure of HO Tyr the soluble precursor
Glycogen procollagen.
for the specificities 1B,
ccountkinase 2 inter-
of the ! synthase
Phosphorylase
containing proteins b with various P –Tyr-
eins. kinase 2
The three to five residues on the !
A Cascade of Proteolytically Activated
nal side of the
Casein P –Tyr
kinase I residue
Atare critical
least nine
theCasein
specificity SH3
! ! ! !
Leads to Blood Coagulation
kinase IIof these 5interactions 0
Pro A blood clot is an aggregate of cell f
Glycogen synthase Ser P
osine-binding domains (PTB domains) platelets, cross-linked and stabilized b
kinase 3 3A, 3B, 3C SH2 ! ! !
nding partner for P –Tyr proteins, but Inactivada
consisting mainly of fibrin (Fig. 6
Autoinhibited
fibers
Glycogen
quences synthase
and three-dimensional structure P derived from a soluble zymogen called f
kinase 4 2
em from SH2 domains. The human !
Tyr albumins and globulins, fibrinogen is ge
 O
OH O P
–

Fosforilación de la bomba sodio-potasio ATPasa

Tyrosine residue Phosphorylated tyrosine residue
O– O

ATP ADP

E E P

2K+ inside 3Na+ inside

Conformational Conformational
change change

2K+ outside 3Na+ outside

E' E' P

dium–
e
binds to P H2O
um.

May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s).
es not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.
 Quinoma y fosfatoma

Cinasas: Todos los genes que codifican para cinasas o quinasas

Se estima que al menos un tercio de todas las proteínas en eucariotes sufren
fosforilación. Muchos eventos de fosforilación son parte de virtualmente todos los
procesos regulatorios.



Fosfatasas:
A la fecha se han encontrado que un total de 107 protein tirosina osfatasas están
codifiadas en el genoma humano
es.indd Page 231 01/09/12 11:38 AM user-F408 /Users/user-F408/Des

Secuencias consenso 6.5 Regulatory Enzymes 231

-Plegamiento tridimencional: acerca resíduos lejanos
Secuencias consenso: carga y entorno básico del reíduo a osforilar
TABLE 6–10 Consensus Sequences for Protein Kinases
Protein kinase Consensus sequence and phosphorylated residue
Protein kinase A -x-R-[RK]-x-[ST]-B-
Protein kinase G -x-R-[RK]-x-[ST]-x-
Protein kinase C -[RK](2)-x-[ST]-B-[RK](2)-
Protein kinase B -x-R-x-[ST]-x-K-
Ca2 !/calmodulin kinase I -B-x-R-x(2)-[ST]-x(3)-B-
Ca2!/calmodulin kinase II -B-x-[RK]-x(2)-[ST]-x(2)-
Myosin light chain kinase (smooth muscle) -K(2)-R-x(2)-S-x-B(2)-
Phosphorylase b kinase -K-R-K-Q-I-S-V-R-
Extracellular signal–regulated kinase (ERK) -P-x-[ST]-P(2)-
Cyclin-dependent protein kinase (cdc2) -x-[ST]-P-x-[KR]-
Casein kinase I -[SpTp]-x(2)-[ST]-B*
Casein kinase II -x-[ST]-x(2)-[ED]-x-
!-Adrenergic receptor kinase -[DE](n)-[ST]-x(3)
Rhodopsin kinase -x(2)-[ST]-E(n)-
Insulin receptor kinase -x-E(3)-Y-M(4)-K(2)-S-R-G-D-Y-M-T-M-Q-I-
G-K(3)-L-P-A-T-G-D-Y-M-N-M-S-P-V-G-D-
Epidermal growth factor (EGF) receptor kinase -E(4)-Y-F-E-L-V-
Sources: Pinna, L.A. & Ruzzene, M.H. (1996) How do protein kinases recognize their substrates? Biochim. Biophys. Acta 1314, 191–225; Kemp, B.E. & Pearson, R.B. (1990) Protein kinase
m the cell surface. In some
vely binding to the surface
e the immune system to at-

Inhibidores de tirosina cinasas

-2006, only eight new drugs
ed States for use in cancer
es and three monoclonal an-
wn efficacy in clinical trials.
ylate (Gleevec; Fig. 3a), one
hibitors, has proved nearly
about remission in patients HN NH
myeloid leukemia. Erlotinib
targets EGF-R, is effective N N O N
l-cell lung cancer (NSCLC). N
N
n signaling systems involve
se, inhibitors that act on sev-
e useful in the treatment of Imatinib (Gleevec)
and sorafenib (Nexavar) tar-
es, including VEGF-R and
are in clinical use for pa-
al stromal tumors and ad-
a, respectively. Trastuzumab N
OH O
Erbitux), and bevacizumab N N
H
al antibodies that target Cl
GF-R, respectively; all three
HO O
certain types of cancer.
ore compounds are in pre- O O OH
drugs being evaluated are
ral sources and some pro- O O N
stry. Indirubin is a compo-
eparation traditionally used Erlotinib (Tarceva) Flavopiridol
it inhibits CDK2 and CDK5. FIGURE 3 Some protein kinase inhibitors now in clinical trials or clini-
nthetic analog of an alkaloid cal use, showing their binding to the target protein. (a) Imatinib binds to
 (Tyr, Ser, Thr, His)
ATP ADP
Modificación covalente reversible
O
B
Enz Enz OPOO!
A
O!
Adenylylation
(Tyr)
ATP PPi O
B
Enz Enz OPOOOCH2 Adenine
A O
O!
H H
H H

OH OH
Acetylation
(Lys, !-amino (amino terminus))
 A O
O!
Modificación covalente reversible
H H
H H

OH OH
Acetylation
(Lys, !-amino (amino terminus))
Acetyl-CoA HS-CoA O
B
Enz Enz OCOCH3

Myristoylation
(!-amino (amino terminus))

Myristoyl-CoA HS-CoA O
B
Enz Enz OCO(CH2)12 OCH3

Ubiquitination
 (!-amino (amino terminus))

Modificación covalente reversible

Myristoyl-CoA HS-CoA
O
B
Enz Enz OCO(CH2)12 OCH3

Ubiquitination
(Lys)
HS- E2 O
O B
U OC U OCOSOE2
O! activation
Activated ubiquitin
O
B
U OCOSOE2
Activated ubiquitin HS- E2 O
B
Enz Enz ONO CO U
H
ADP-ribosylation
 Sistema Ubiquitina-Proteosoma MECHANISMS OF DISEASE

Ubiquitin conjugation Protein degradation

26S
Proteasome
Ub
Ub Amino
Ub
Ub acids
Ubiquitin Ub
Ub Ub
Ub
Ub
Ub
Ub
Ub Ub
Ub Ub
Ub
E1, E2, E3 Ub
Ub
Ub
Ub
ATP ATP

Peptides
Protein ADP

Antigen
ATP presentation

19S complex
B

20S core proteasome

Figure 3. The Ubiquitin–Proteasome Pathway of Proteolysis.

teins in
at their O
B
as a tag U OCOSOE2

Modificación covalente reversible

on (see
gulatory
Activated ubiquitin HS- E2 O
B
Enz Enz ONO CO U
karyotic H
of tran-
ADP-ribosylation
(Arg, Gln, Cys, diphthamide—a modified His)
ng reac-
NAD nicotinamide
ribose is
(NAD) Enz Enz
s for the
ulting in O O

U
nitrogen H2CO O O P O O O P OO O CH2 O
enzymes Adenine A A
O O !
O! H H
tion) of H H
H H
H H OH OH
portant
hat one- OH OH
sphory- Methylation
Áci. glutámico (Glu)
n events
me pro- S-adenosyl- S-adenosyl-
methionine homocysteine
ers have
phoryla- Enz EnzOCH3
tral to a
tein specific to liver (Fig. 15–13). The binding is much pathway. The met
tighter in the presence of the allosteric effector fructose 6- alyzed by PFK-1 is
Secuestro nucelar y proteína reguladora
phosphate. Glucose competes with fructose 6-phosphate
for binding and causes dissociation of the regulatory protein
colysis. In addition
complex enzyme h
from the hexokinase, relieving the inhibition. Immediately allosteric activators

Capillary
Cytosol Nucleus
GLUT2
Glucose
Glucose

Plasma Hexokinase IV Hexokinase IV

membrane

Regulatory
Glucose 6-phosphate protein

Fructose 6-phosphate
FIGURE 12–5 Self-inactivation of Gs. The steps are further described in
residues in this cleft region have
the text. The protein’s intrinsic GTPase activity, in many cases stimu- all of the more than 1,000 known
lated by RGS proteins (regulators of G-protein signaling), determines As indicated in Figure 12–

PKA dependiente de cAMP

how quickly bound GTP is hydrolyzed to GDP and thus how long the
G protein remains active.
lates several enzymes downstre
way (Table 12–2). Although th

(a) (b) Substrate

AKAP
Dimerization Inhibitor sequence binding cl
Substrate- domain
Small lobe
binding cleft (ATP binding)

Inhibitor
Catalytic sequence
subunit C CC
R R
Regulatory
subunit
4 cAMP 4 cAMP

Regulatory
(R) subunit
Substrate-binding
cleft now available

2 cAMP 2 cAMP
ase Gluconeogenesis
phatase (catalytic subunit) Glucose release to blood

o enzymes of gluconeoge-
d glucose 6-phosphatase
Inducción génica
Glucose
Plasma
mportant to carbohydrate GLUT2 membrane
ohydrate response ele-
5–21), which is expressed Cytosol
e, and kidney. It serves to Glucose
ymes needed for carbohy- hexokinase IV
(glucokinase)
BP in its inactive state is
in the cytosol. When the Glucose
6-phosphate
P2A (Fig. 15–18) removes
EBP, the transcription fac-
e, nuclear PP2A removes P P
ChREBP now joins with a Xylulose
ChREBP
5-phosphate
on the synthesis of several
atty acid synthase, and PP2A
Pi
rst enzyme in the path to P
).
ChREBP

egulation by the transcription P Nucleus

Xylulose
cytosol of a hepatocyte is phos- ChREBP
5-phosphate
it cannot enter the nucleus. De-
ein phosphatase PP2A allows PP2A
Pi
a second dephosphorylation, of
can associate with its partner Mlx
Ml BP

Ml BP

ChREBP mRNA
RE

RE

s to the carbohydrate response

x

x
Ch

Ch

stimulates transcription. PP2A is

hosphate, an intermediate in the ChoRE
gulation
Inducción génica
PP2A—and thus, ultimately,
metabolic enzymes—is xylu- Insulin
diate not of glycolysis or glu-
entose phosphate pathway.
Plasma
ation is high, glucose enters membrane
d by hexokinase IV. The glu-
ed can enter either the gly- Cytosol
se phosphate pathway. If the
oduce xylulose 5-phosphate,
he glucose-utilizing pathways Proteasome
te. It accomplishes this by al-
PKB Ubiquitin
which then dephosphorylates
ption factor to turn on the ex-
of glycolysis and fat synthe- FOXO1 Pi FOXO1 P
lds pyruvate, and conversion
ovides the starting material phosphoprotein
phosphatase
yl-CoA carboxylase converts
he first committed intermedi- Nucleus
The fatty acid synthase com-
export to adipose tissue and FOXO1
apter 21). In this way, excess DNA
mRNA
as fat.
actor in the liver, SREBP- PEP carboxykinase
Glucose 6-phosphatase
of sterol response ele-
e Fig. 21–43), turns on the
, hexokinase IV, lipoprotein FIGURE 15–22 Mechanism of gene regulation by the transcription
factor FOXO1. Insulin activates the signaling cascade shown in Figure
 CAPÍTULO 9 Enzimas: regulación de actividades 91

los cuales se
Fosforilación-desfodforilación
CUADRO 9–1 Ejemplos de enzimas de mamífero
ral lo están, cuya actividad catalítica es alterada por
fosforilación-desfosforilación covalente
Estado de actividad

Enzima Bajo Alto

Acetil-CoA carboxilasa EP E
e proteína y
e otra forma Glucógeno sintasa EP E
, el “ligando Piruvato deshidrogenasa EP E
Tanto la fos-
HMG-CoA reductasa EP E
retroacción
eversible, del Glucógeno fosforilasa E EP
gicas especí- Citrato liasa E EP
. Las dos ac-
Fosforilasa b cinasa E EP
a metabólica
úan en sitios HMG-CoA reductasa cinasa E EP
hibición por
Abreviaturas: E, desfosfoenzima; EP, fosfoenzima.
e caracterís-
ción de enzi-
n comprende una respuesta celular coordinada apropiada. En estas redes re-
 Activación proteolítica de Zimógenos
Zimógenos:

-Proteasas sintetizadas de forma inactiva o como proenzimas

-Evitar daño en el órgano que la sintetetiza

-Están disponibles para su activación rápida

-Su activación es irreversible

-Deben haer proteínas reguladoras

-Proteasas digestivas, enzimas de la coagulación, sistema del complemento

-Ruptura específica causa cambio conformacional que expone el sitio activo de la enzima
e. For example, activate multiple copies of the next prote
0) binds to and the signal is amplified in each step. In s
Activación proteolítica de Zimógenos
Trypsinogen
(inactive)
1 6 7 229
Lys–Ile
Inhibidor pancreático de tripsina
enteropeptidase

Val1–(Asp)4 –Lys6

Trypsin
(active)
7 229
Ile
very tightly to the enzyme active site. For example, activate multiple copies of the
pancreatic trypsin inhibitor (Mr 6,000) binds to and the signal is amplified in each
Activación proteolítica de Zimógenos
Chymotrypsinogen Trypsinogen
(inactive) (inactive)
1 245 1 6 7 229
Lys–Ile
enteropeptidase
trypsin
Val1–(Asp)4 –Lys6

Carboxypeptidases A and B
p -Chymotrypsin Trypsin
(active) (active)
1 15 16 245 7 229
Inhibidor pancreático de tripsina:
Arg Ile Ile

p -chymotrypsin Prevención de activación prematura de

(autolysis)
zimógenos. FIGURE 6–38 Activation of zymogens by pro
the formation of chymotrypsin and trypsin f
Ser14–Arg15
! Thr147–Asn148 sinogen and trypsinogen. The bars represe
α1- antiproteinasa: inhibidor de la elastasa de
the polypeptide chains, with numbers indica
a -Chymotrypsin neutrofilos. Defectos: enfisema, daño hepáico
(the amino-terminal residue is number 1). Re
(active) peptide fragments generated by cleavage are i
1 13 16 146 149 245 Factores de coagulación
in the final active forms, some numbered res
Leu Ile Tyr Ala three polypeptide chains (A, B, and C) of ch
A B C bonds (see Fig. 6–19).
Glucógeno fosforilasa
 has persisted in common usage and in the literature.) glucose 1-phos
The enzyme (phosphorylase b kinase) responsi- are adequate,
ble for activating phosphorylase by transferring a phos-
Activación de la glucógeno fosforilasa
AMP binds, ina
phoryl group to its Ser residue is itself activated by When the
phosphorylas
protein phosp
Ser14 OH OH Ser14
side side ryl groups from
chain CH2 CH2 chain less active form
Like the e
Phosphorylase b
(less active) phorylase of l
phorylation/de
dephosphoryla
glucagon
(liver)
blood glucose
2Pi 2ATP the cascade m
phosphorylase a phosphorylase b
phosphatase kinase
(PP1)
2H2O 2ADP epinephrine, FIGURE 15–34 Reg
[Ca2+], [AMP]
(muscle) lent modification.
lase a, Ser14 resi
P P
Phosphorylase a is
O O
by enzymatic loss
CH2 CH2
rylase a phosphata
Phosphorylase a PP1). Phosphoryla
(active) lase a by the actio
glycogen phospho
 m of epinephrine and
Epinephrine x molecules Glucagon
ecific surface receptors, Myocyte Hepatocyte
ocyte (left) or glucagon
ates a GTP-binding pro-
riggers a rise in [cAMP],
de of phosphorylations; Gs!
se, which then activates
cades effect a large am-
figures in pink boxes are
ATP Cyclic AMP
al increase in number of adenylyl
cyclase 20x molecules
ade. The resulting break-
e, which in the myocyte
muscle contraction and Inactive PKA Active PKA
he blood to counter the
10x molecules
[Ca2+]
Inactive Active
phosphorylase b phosphorylase b
kinase kinase
100x molecules
Inactive Active
glycogen glycogen
phosphorylase b phosphorylase a

[AMP] 1,000x molecules

Glycogen Glucose 1-phosphate

10,000x molecules
 Importancia

ü  Mecanismo de acción de muchos fármacos

ü  Se utilizan como medicamentos. Ejemplo: L-asparaginasa

ü  Blanco de la acción de algunso venenos: cianuro

ü  Las enzimas como reactivos en determinaciones de laboratorio

ü  Deficiencia o ausencia total de una o varias enzimas en varios desordenes
genéticos hereditarios (errores congénitos del metabolismo)

ü  Herramientas en ingenieria química, tecnología de alimentos y agricultura

 denomina comúnmente transaminasas y su otra
de
Clasificación Clinica de las enzimas
División de las enzimas que aparecen es
qu
en el plasma según su origen
Grupo Ejemplos es
Protrombina, qu
plasminógeno, po
Especificas del plasma ceruloplasmina, sa
o Funcionales de plasma lipoproteinlipasa, ce
pseudocolinstearasa
Amilasa pancreática, salival, T
De secreción fosfatasa prostática, So
pepsinógeno. tr
LDH; MDH; Glicerol-3- am
De vías fosfato Deshidrogenasa; 1,6 co
Enzimas principales difosfo-fructo-aldolasa; el
celulares GOT; GPT. am
Ez del ciclo de la urea; E
Organoespecificas re
glucosa-6-fosfatasa; 5´ND.
tr
Enzimas y proteínas en infarto de miocardio

Corazón das de maner

teria pueden
Arteria coronaria factores de cr
ej., factor de
Trombo acumulan pr
no y calcio e
de LDL oxid
Infarto ticular impor
crófagos (cél
Liberación de
troponinas, CK-MB,
de crecimien
Mb, AST, ALT, LDH-1 en la ateroscl
y otras proteínas y crófagos y lin
enzimas, hacia la na C reactiva
circulación
crónica. A m
tría adiposa e
FIGURA 57–19 Diagrama de un trombo en una arteria ber inflamaci
 Myoglobin (FD
Myoglobin is a heme-containing protein that binds oxygen within cardiac my
and skeletal muscle; only a single form is common to both muscle types. 20
Enzimas y proteínas en infarto de miocardio
Having a molecular weight of only 18 kDa, myoglobin apparently leaks affi
from damaged cells more rapidly than other proteins. Following MI, a s
ari
em
Myoglobin, or bef
CK-MB isoform ratio to
cle
Transaminase, total CK,
sen
or CK-MB tha
100
Marker concentration

LD-1 con
(% of maximum)

80 on
at
60 cTnI pre
est
40 or cTnT
20 AS
0 Th
0 24 48 72 96 120 144
tot
Hours after onset of chest pain sep
cT
Figure 18-1 Schematic depiction of the kinetics of several cardiac markers fol-
asa. paciente, sexo, antecedentes personales no patológicos y patoló-

Priincipales enzimas utilizadas en el

gicos, posible consumo de fármacos o drogas, así como la sensi-
bilidad y la especificidad diagnóstica de la prueba enzimática.
S ENZIMAS
CO
CUADRO diagnóstico clínico
7–2 Principales enzimas séricas usadas
en el diagnóstico clínico
íneo ha desempeñado Enzima sérica Principal uso diagnóstico
co de varios procesos
Aminotransferasas
entes funcionales de la
docolinesterasa, lipo- Aspartato aminotransferasa Infarto de miocardio
(AST o SGOT)
Alanina aminotransferasa (ALT Hepatitis viral
o SGPT)

P, Mg2+ Amilasa Pancreatitis aguda

Ceruloplasmina Degeneración hepatolenticular

(enfermedad de Wilson)
DP, Mg2+
Creatina cinasa Trastornos musculares e infarto
de miocardio
ADP+
γ-Glutamil transferasa Diversas enfermedades hepáticas

Isozima 5 de la lactato Enfermedades hepáticas

ADPH + H+
deshidrogenasa
na
Lipasa Pancreatitis aguda
oplada para la actividad
sfato mediante la Fosfatasa ácida Carcinoma metastásico
de la próstata
producto por la glucosa-
ma añadida y NADP+. Fosfatasa alcalina (isozimas) Diversos trastornos óseos,
shidrogenasa, el índice enfermedades hepáticas obstructivas
340 nm, está regido por
 Uso de esterasas en química y farmcia

En la industria farmacéutica las esterasas se usan para obtener fármacos ópticamente

puros o precursores de los mismos a partir de mezclas racémicas (bornscheuer 2002).
Probablemente la esterasa más conocida es la llamada carboxil esterasa NP (por el
naproxeno), aislada de B. Subtilis (quax y broekhuizen 1994) que se utiliza para la
resolución de (R, s)-naproxeno y de (r,s)-ibuprofeno metilester con una excelente pureza
óptica. Para la síntesis de ibuprofeno también se utiliza la esterasa de pseudomonas sp.
(Kim et al. 2002a; kim et al. 2002b). Además, la esterasa de trichosporon brassicae se
usa en la producción del fármaco antiinflamatorio ketoprofeno (wu et al. 2003).
 Industra alimentaria

Un ejemplo prominente del uso de carboxil esterasas para la industria alimentaria es la

liberación del ácido ferúlico de la pared polisacarídica de plantas. El ácido ferúlico es
precursor de la vainilla, un saborizante ampliamente utilizado y de alto valor comercial
(Gasson et al. 1998).

 Uso de esterasas en agricultura

En agricultura se han utilizado las fosfodiesterasas para sintetizar compuestos

organofosforados como el cumafos y sus oxoderivados, ampliamente usados como
insecticidas y nematicidas. Los residuos de estos compuestos son tóxicos
ambientales que pueden contaminar productos alimenticios. En este contexto, las
fosfodiesterasas de brevundimonas diminuta y alteromonas sp. Han sido usadas en
detoxificación y degradación de este tipo de compuestos organofosforados
(horne et al. 2002)
 Referencias
One way in which this condition might be fulfilled would be if the
molecules when combined with the enzyme, lay slightly further apart
than their equilibrium distance when [covalently joined], but nearer
than their equilibrium distance when free. . . . Using Fischer’s lock and
key simile, the key does not fit the lock quite perfectly but exercises a
certain strain on it.
6
—J. B. S. Haldane, Enzymes, 1930

Catalysis can be described formally in terms of a stabilization of the

transition state through tight binding to the catalyst.
—William P. Jencks, article in Advances in Enzymology, 1975

Enzymes
Enzimas, capítulo 6, Lehninger Principios de Bioquímica, 5ª edición, H. Freeman and company
6.1 An Introduction to Enzymes 183 They have a high degree of specificity for their sub-
strates, they accelerate chemical reactions tremen-
New York, 2008 , páginas 183 a 228.
6.2 How Enzymes Work 186 dously, and they function in aqueous solutions under
very mild conditions of temperature and pH. Few nonbi-
6.3 Enzyme Kinetics as an Approach to Understanding ological catalysts have all these properties.
Mechanism 194 Enzymes are central to every biochemical process.
6.4 Examples of Enzymatic Reactions 205 Acting in organized sequences, they catalyze the hun-
dreds of stepwise reactions that degrade nutrient mole-
6.5 Regulatory Enzymes 220 cules, conserve and transform chemical energy, and
make biological macromolecules from simple precursors.
The study of enzymes has immense practical impor-

T here are two fundamental conditions for life. First, tance. In some diseases, especially inheritable genetic
the organism must be able to self-replicate (a topic disorders, there may be a deficiency or even a total
considered in Part III); second, it must be able to absence of one or more enzymes. Other disease condi-
 Referencias

Peter J. Kennelly, PhD y Victor W. Rodwell, PhD. Enzimas: mecanismo de acción, Harper
Bioquímica ilustrada, 29a. edición, México, D.F. McGRAW-HILL INTERAMERICANA EDITORES,
S.A. de C.V. 2012. páginas 57 a 68
 Referencias

Peter J. Kennelly, PhD y Victor W. Rodwell, PhD. Enzimas: cinética, Harper Bioquímica
ilustrada, 29a. edición, México, D.F. McGRAW-HILL INTERAMERICANA EDITORES, S.A. de C.V.
2012. páginas 70 a 83
 Referencias

Peter J. Kennelly, PhD y Victor W. Rodwell, PhD. Enzimas: regulación de actividades, Harper
Bioquímica ilustrada, 29a. edición, México, D.F. McGRAW-HILL INTERAMERICANA EDITORES,
S.A. de C.V. 2012. páginas 84 a 93