Chapter 2
Enzyme
Contents
1. Properties of enzymes
2. Structural features of enzymes
3. Mechanism of enzyme-catalyzed reactions
4. Kinetics of enzyme-catalyzed reactions
5. Inhibition of enzymes
6. Regulation of enzymes
7. Clinical applications of enzymes
8. Nomenclature
Section 1
Properties of Enzymes
§ 1.1 General Concepts
A+B→C+D
[C][D]
GG RTln
[A][B]
0
• spontaneous reaction only if G is negative.
• at equilibrium if G is zero.
• spontaneously impossible if G is positive.
Free energy
transition state, S
G+ (uncatalyzed)
reactants
G for
the reaction
products
Reaction progress
Catalyzed reactions
• Reactants need to pass over the energy
barrier, G+.
• Catalysts reduce the activation energy
and assist the reactants to pass over the
activation energy.
Free energy
transition state, S
G+ (uncatalyzed)
G+ (catalyzed)
reactants
G for
the reaction
products
Reaction progress
Need for special catalysts
Chemical reactions in living systems are
quite different from that in the industrial
situations because of
1. Fragile structures of the living systems
2. Low kinetic energy of the reactants
3. Low concentration of the reactants
4. Toxicity of catalysts
5. Complexity of the biological systems
Enzymes
• Enzymes are catalysts that have special
characteristics to facilitate the
biochemical reactions in the biological
systems.
• Enzyme-catalyzed reactions take place
usually under relatively mild conditions.
§ 1.2 Characteristics
Enzyme-catalyzed reactions have the
following characteristics in comparison
with the general catalyzed reactions:
• common features: 2 “do” and 2 “don’t”
• unique features: 3 “high”
Common features
• Do not consume themselves: no
changes in quantity and quality before
and after the reactions.
• Do not change the equilibrium points:
only enhance the reaction rates.
• Apply to the thermodynamically
allowable reactions
• Reduce the activation energy
Unique features
• Enzyme-catalyzed reactions have very
high catalytic efficiency.
• Enzymes have a high degree of
specificity for their substrates.
• Enzymatic activities are highly regulated
in response to the external changes.
§ 1.3.a High efficiency
Catalyst
Activation energy
(cal/M)
No catalyst
18,000
Normal catalyst
11,700
Hydrogen peroxidase
2,000
Accelerated reaction rates
enzyme
Non-enzymatic
rate constant
(kn in s-1)
enzymatic
rate constant
(kn in s-1)
accelerated
reaction rate
Carbonic anhydrase
10-1
106
8 x 106
Chymotrypsin
4 x 10-9
4 x 10-2
10-2107
Lysozyme
3 x 10-9
5 x 10-1
2 x 108
Triose phosphate
isomerase
4 x 10-6
4 x 103
109
Urease
3 x 10-10
3 x 104
1014
Mandelate racemase
3 x 10-10
5 x 102
1.7 x 1015
10-15
102
1017
Alkaline
phosphatase
§ 1.3.b High specificity
Unlike conventional catalysts, enzymes
demonstrate the ability to distinguish
different substrates. There are three types
of substrate specificities.
• Absolute specificity
• Relative specificity
• Stereospecificity
Absolute specificity
Enzymes can recognize only one type of
substrate and implement their catalytic
functions.
urease
NH2
O
+ H 2O
C
NH2
urea
NH CH3
O
C
+ H2O
NH2
methyl urea
2NH3 + CO2
Relative specificity
Enzymes catalyze one class of substrates
or one kind of chemical bond in the same
type.
protein kinase A
protein kinase C
protein kinase G
To phopharylate the -OH group of serine
and threonine in the substrate proteins,
leading to the activation of proteins.
sucrose
CH2OH
O H
H
H
1
OH H
OH
H
OH
1
CH2OH
O
O
H
H
OH
CH2OH
OH
H
CH2OH
sucrase
O H
OH
H
1
1
OH H
O CH2
CH2OH
H
O
O H
H
H
H
OH
H
1
OH
H
OH H
O
CH2OH
raffinose
OH
H
H
OH
OH
Stereospecificity
The enzyme can act on only one form of
isomers of the substrates.
H3C
A
H
H
C
OH
C
COOH
OH
B
COOH
C
H3C
A
B
C
Lactate dehydrogenase can recognize only the
L-form but the D-form lactate.
§ 1.3.c High regulation
• Enzyme-catalyzed reactions can be
regulated in response to the external
stimuli, satisfying the needs of biological
processes.
• Regulations can be accomplished
through varying the enzyme quantity,
adjusting the enzymatic activity, or
changing the substrate concentration.
Section 2
Components of Enzymes
§ 2.1 Active Center
• Almost all the enzymes are proteins
having well defined structures.
• Some functional groups are close
enough in space to form a portion called
the active center.
• Active centers look like a cleft or a
crevice.
• Active centers are hydrophobic.
Lysozyme
Residues (colored ) in the active site come from
different parts of the polypeptide chain .
Two essential groups
The active center has two essential groups
in general.
• Binding group: to associate with the
reactants to form an enzyme-substrate
complex
• Catalytic group: to catalyze the
reactions and convert substrates into
products
Protein chain
Substrate
molecule
Essential groups
outside the
active center
+
-
Catalytic group
Active center
Binding group
Active centers
§ 2.2 Molecular Components
• Simple enzymes: consists of only one
peptide chain
• Conjugated enzymes:
holoenzyme = apoenzyme + cofactor
(protein)
(non-protein)
• Cofactors: metal ions; small organic
molecules
Metal ions
• Metal-activated enzyme: ions necessary
but loosely bound. Often found in metalactivated enzyme.
• Metalloenzymes: Ions tightly bound.
• Particularly in the active center, transfer
electrons, bridge the enzyme and
substrates, stabilize enzyme
conformation, neutralize the anions.
Organic compounds
• Small size and chemically stable
compounds
• Transferring electrons, protons and other
groups
• Vitamin-like or vitamin-containing
molecule
Coenzymes
• Loosely bind to apoenzyme. Be able to
be separated with dialysis.
• Accepting H+ or group and leaving to
transfer it to others, or vise versa.
Prosthetic groups
• Tightly bind through either covalent or many
non-covalent interactions.
• Remained bound to the apoenzyme during
the course of reaction.
Section 3
Mechanism of EnzymeCatalyzed Reactions
To understand the molecular details of the
catalyzed reaction.
• Proximity and orientation arrangement
• Multielement catalysis
• Surface effect
Lock-and-key model
Both E and S are rigid and fixed, so they must be
complementary to each other perfectly in order to
have a right match.
Induced-fit model
The binding induces conformational changes of
both E and S, forcing them to get a perfect
match.
Hexokinase catalyzing glycolysis
• Hexokinase, the first enzyme in the
glycolysis pathway, converted glucose
to glucose-6-phosphate with consuming
one ATP molecule.
• Two structural domains are connected
by a hinge.
• Upon binding of a glucose molecule,
domains close, shielding the active site
for water.
Induced structural changes
Section 4
Kinetics of EnzymeCatalyzed Reactions
§ 4.1 Reaction rate
[P]
[P]
Initial slope = vo =
t
0
Time (t)
[P]
t
Initial velocity
• The reaction rate is defined as the
product formation per unit time.
• The slope of product concentration ([P])
against the time in a graphic
representation is called initial velocity.
• It is of rectangular hyperbolic shape.
Reaction velocity curve
V0
Vmax
Vmax/2
[S]
0
Km
Intermediate state
Forming an enzyme-substrate complex,
a transition state, is a key step in the
catalytic reaction.
k1
E + S
ES
k3
E + P
k2
initial
intermediate
final
Free energy
transition state, S
G+ (uncatalyzed)
G+ (catalyzed)
reactants
G for
the reaction
products
Reaction progress
Rate constants
k1
E + S
ES
k3
E + P
k2
• K1 = rate constant for ES formation
• K2 = rate constant for ES dissociation
• K3 = rate constant for the product
released from the active site
§ 4.2 Michaelis-Menten Equation
• The mathematical expression of the
product formation with respect to the
experimental parameters
• Michaelis-Menten equation describes
the relationship between the reaction
rate and substrate concentration [S].
Assumptions
• [S] >> [E], changes of [S] is negligible.
• K2 is negligible compared with K1.
• Steady-state: the rate of E-S complex
formation is equal to the rate of its
disassociation (backward E + S and
forward to E + P)
[S]
V = K
V
max
m + [S]
Describing a hyperbolic curve.
Km is a characteristic constant of E
[S] << Km 时，v ∝ [S]
[S] >> Km 时，v ≈ Vmax
V0
Vmax
Zero order with
respect to [S]
First order with
respect to [S]
0
[S]
Significance of Km
• the substrate concentration at which
enzyme-catalyzed reaction proceeds at
one-half of its maximum velocity
• Km is independent of [E]. It is
determined by the structure of E, the
substrate and environmental conditions
(pH, T, ionic strength, …)
V0
Vmax
Vmax/2
[S]
0
Km
• Km is a characteristic constant of E.
• The value of Km quantifies the affinity of
the enzyme and the substrate under the
condition of K3 << K2. The larger the Km，
the smaller the affinity.
k2 + k3
Km =
k1
Km for selected enzymes
Enzyme
Substrate
km
Catalase
H2O2
25
Hexokinase
ATP
0.4
D-Glucose
0.05
D-Fructose
1.5
Carbonic anhydrase
HCO3-
Chemotrypsin
Glycyltyrosinylglycine
108
N-Benzoyltyrosinamide
2.5
9
Galactosidase
D-Lactose
4
Threonine dehydratase
L-Threonine
5
Significance of Vmax
• The reaction velocity of an enzymatic
reaction when the binding sites of E are
saturated with substrates.
• It is proportional to [E].
Turnover number
k3 = Vmax / [E]
• Vmax is the reaction rate when the
enzymes are saturated, and is
independent of the enzyme
concentration.
• The number of the products converted
in a unit time by one enzyme molecule
which is saturated.
Lineweaver-Burk plot
Km 1
1
1
+
=
Vmax [S]
Vmax
V
• To determine Km and Vmax
• To identify the reversible repression
Double-reciprocal plot
1/V
Slope = Km/Vmax
Intercept = -1/ Km
Intercept = 1/Vmax
1/[S]
§ 4.3 Factors affecting enzymecatalyzed reaction
• Substrate concentration
• Enzyme concentration
• Temperature
• pH
• Inhibitors
• Activators
§ 4.3.a Effect of substrate
• Has been described already.
• [E] affects the rate of
enzyme-catalyzed
reactions
• [S] is held constant.
Reaction velocity
§ 4.3.b Effect of enzyme
• When [S] >> [E], V ≈
[E]
Enzyme concentration
§ 4.3.c Effect of temperature
• Optimal temperature (To) is the
characteristic T at which an enzyme has
the maximal catalytic power.
• 35 ~ 40C for warm blood species.
• Reaction rates increase by 2 folds for
every 10C rise.
• Higher T will denature the enzyme.
Enzymatic activity
2.0
1.5
1.0
0.5
10
20
30
40
50
60
Temp. (C)
§ 4.3.d Effect of pH
• Optimal pH is the characteristic pH at
which the enzyme has the maximal
catalytic power.
• pH7.0 is suitable for most enzymes.
• Particular examples:
pH (pepsin) = 1.8
pH (trypsin) = 7.8
Enzymatic activity
2.0
pepsin
1.5
trypsin
1.0
0.5
2.0
4.0
6.0
8.0
10.0
pH
Section 5
Inhibition of Enzyme
§ 5.1 Inhibitors
• Inhibitors are certain molecules that can
decrease the catalytic rate of an
enzyme-catalyzed reaction.
• Inhibitors can be normal body
metabolites and foreign substances
(drugs and toxins).
Inhibition processes
• The inhibition process can be either
irreversible or reversible.
• The inhibition can be competitive, noncompetitive, or un-competitive.
§ 5.2 Irreversible inhibition
• Inhibitors are covalently bound to the
essential groups of enzymes.
• Inhibitors cannot be removed with
simple dialysis or super-filtration.
• Binding can cause a partial loss or
complete loss of the enzymatic activity.
Pesticide poisoning
choline esterase
acetylcholine
choline + acetic acid
Acetylcholine accumulation will cause
excitement of the parasympathetic system:
omitting, sweating, muscle trembling, pupil
contraction
+ E OH
P
R'O
RO
X
RO
+
P
O
organophosphate
O E
R'O
AChE
O
acid
inhibited AChE
+
N
HX
CHNOH
CH3
E OH
PAM
O
OR'
P
+
N
CH3
CHNO
OR
Heavy metal poisoning
• Heavy metal containing chemicals bind to the
–SH groups to inactivate the enzymes.
S
SH
+
E
Hg2+
SH
2H+
S
Cl
+
E
+
S
SH
SH
Hg
E
Cl
As C
H
CHCl
E
S
As C
H
CHCl
+
2HCl
§ 5.3 Reversible inhibition
• Inhibitors are bound to enzymes noncovalently.
• The reversible inhibition is characterized
by an equilibrium between free enzymes
and inhibitor-bound enzymes.
§ 5.3.a Competitive inhibition
• Competitive inhibitors share the
structural similarities with that of
substrates.
• Competitive inhibitors compete for the
active sites with the normal substrates.
• Inhibition depends on the affinity of
enzymes and the ratio of [E] to [S].
Vmax [S]
V=
[I]
Km(1 +
1
V
=
Km
Vmax
Ki
) + [S]
[I]
(1 +
Ki
1
1
)
+
Vmax
[S]
Lineweaver-Burk plot
Competitive
inhibitor
increase
1/V
1/Vmax
No inhibitor
-1/ Km
1/[S]
-1/ Km(1 + [I]/Ki)
Inhibition features
• As [S] increases, the effect of inhibitors
is reduced, leading to no change in Vmax.
• Due to the competition for the binding
sites, Km rises, equivalent to the
reduction of the affinity.
Example-1: competitive inhibitor
• FH4 (tetrahydrofolate) is a coenzyme in the
nucleic acid synthesis, and FH2 (dihydrofolate)
is the precursor of FH4.
• Bacteria cannot absorb folic acid directly from
environment.
• Bacteria use p-amino-benzoic acid (PABA),
Glu and dihydropterin to synthesize FH2.
• Sulfanilamide derivatives share the structural
similarity with PABA, blocking the FH2
formation as a competitive inhibitor.
Glu
+
COOH
H2N
FH2
FH2
synthetase
PABA
FH4
FH2
reductase
+
dihydropterin
H2N
SO2NHR
Sulfanilamide
Methotrexate
Example-2: competitive inhibitor
COOH
COOH
succinate
dehydrogenase
H2C
HC
CH
CH2
HOOC
HOOC
succinate
fumaric acid
CO-COOH
COOH
H2C
H2C
COOH
COOH
malonic acid
oxaloacetate
§ 5.3.b Non-competitive inhibition
• Inhibitors bind to other sites rather than
the active sites on the free enzymes or
the E-S complexes.
• The E-I complex formation does not
affect the binding of substrates.
• The E-I-S complexes do not proceed to
form products.
• Reducing the [E-S]
• Vmax↓; unchanged Km.
noncompetitive
inhibitor
increase
1/V
(1 + [I]/Ki)/Vmax
No inhibitor
-1/ Km
1/[S]
1
V
=
Km
Vmax
[I]
(1 +
Ki
[I]
1
1
)
(1 +
)
+
Vmax
[S]
Ki
§ 5.3.c Uncompetitive inhibition
• Uncompetitive inhibitors bind only to the
enzyme-substrate complexes.
• The E-I-S complexes do not proceed to
form products.
• The E-I-S complexes do not backward to
the substrates and enzymes.
• This inhibition has the effects on
reducing both Vmax and Km.
• Commonly in the multiple substrate
reactions.
Uncompetitive
inhibitor
increase
1/V
(1 + [I]/Ki)/Vmax
No inhibitor
1/Vmax
-1/ Km
1/[S]
-1/ Km(1 + [I]/Ki)
1
V
=
Km
Vmax
[I]
1
1
)
(1 +
+
Vmax
[S]
Ki
Summary of inhibition
type
binding target
Km
Vmax
Competitive
E only

=
Noncompetitive
E or ES
=

Uncompetitive
ES only


Activator
Activators are the compounds which bind
to an enzyme or an enzyme-substrate
complex to enhance the enzymatic
activity without being modified by the
enzymes.
Activators
• Metal ions
• essential activators: no enzymatic
activity without it
Mg2+ of hexokinase
• non-essential activators: enhancing the
catalytic power.
Enzymatic activity
• Enzymatic activity is a measure of the
capability of an enzyme of catalyzing a
chemical reaction.
• It directly affects the reaction rate.
• International unit (IU): the amount of
enzyme required to convert 1 µmol of
substrate to product per minute under a
designated condition.
• Determination of the enzymatic activity
requires proper treatment of enzymes,
excess amount of substrate, optimal T
and pH, …
• One katal is the amount of enzyme that
converts 1 mol of substrate per second.
• IU = 16.67×10-9 kat
§ 2.2 Molecular Components
• In addition to enzymes, other chemical
species often participate in the catalysis.
• Cofactor: chemical species required by
inactive apoenzymes (protein only) to
convert themselves to active holoenzymes.
Cofactors
Cofactors
Essential ions
Activator ions
(loosely bound)
Metal ions of
metalloenzymes
(tightly bound)
Coenzymes
Cosubstrates
(loosely bound)
Prosthetic
groups
(tightly bound)
Essential ions
• Activator ions: loosely and reversibly
bound, often participate in the binding of
substrates.
• Metal ions of metalloenzymes: tightly
bound, and frequently participate directly
in catalytic reactions.
Function of metal ions
• Transfer electron
• Linkage of S and E；
• Keep conformation of E-S complex
• Neutralize anion
Coenzymes
• Act as group-transfer reagents to supply
active sites with reactive groups not
present on the side chains of amino
acids
• Cosubstrates:
• Prosthetic groups:
Cosubstrates
• The substrates in nature.
• Their structures are altered for subsequent
reactions.
• Shuttle mobile metabolic groups among
different enzyme-catalyzed reactions.
Prosthetic groups
• Supply the active sites with reactive
groups not present on the side chains of
AA residues.
• Can be either covalently attached to its
apoenzymes or through many noncovalent interactions.
• Remained bound to the enzyme during
the course of the reaction.
Coenzymes
• Metabolite coenzymes: they are
synthesized from the common
metabolites.
– several NTP, ATP (most abundant), UDPglucose
• Vitamin-derived coenzymes: they are
derivatives of vitamins, and can only be
obtained from nutrients.
– NAD and NADP+, FAD and FMN, lipid
vitamins, …
§ 2.3 Ribozyme
• Until recently, all the enzymes are
known to be proteins.
• Ribonucleic acids also demonstrate the
catalytic ability.
• Ribozymes have the ability to selfcleave.
• They are highly conservative, an
indication of the biological evolution and
the primary enzyme.
Family of serine protease
Hydrolysis
site
Trypsin
Hydrolysis
site
Chymotrypsin
Hydrolysis
site
Elastase
§ 5.3.a Competitive inhibition
E+S
+
I
Ki
EI
ES
E+P
§ 5.3.b Non-competitive inhibition
E+S
+
I
ES
+
I
Ki
Ki
EI + S
EIS
E+P
§ 5.3.c Uncompetitive inhibition
E+S
ES
+
I
Ki
EIS
E+P
Section 6
Regulation of Enzyme
• Many biological processes take place at a
specific time; at a specific location and at a
specific speed.
• The catalytic capacity is the product of the
enzyme concentration and their intrinsic
catalytic efficiency.
• The key step of this process is to regulate
either the enzymatic activity or the enzyme
quantity.
Reasons for regulation
• Maintenance of an ordered state in a timely
fashion and without wasting resources
• Conservation of energy to consume just
enough nutrients
• Rapid adjustment in response to
environmental changes
Controlling an enzyme that catalyzes the
rate-limiting reaction will regulate the entire
metabolic pathway, making the biosystem
control more efficient.
Rate limiting reaction is the reaction whose
rate set by an enzyme will dictate the whole
pathway, namely, the slowest one or the
“bottleneck” step.
§6.1 Regulation of E Activity
• Zymogen activation
• Allosteric regulation
• Covalent modification
§6.1.a Zymogen activation
• Certain proteins are synthesized and secreted
as an inactive precursor of an enzyme, called
zymogen.
• Selective proteolysis of these precursors leads
to conformational changes, and activates these
enzymes.
• It is the conformational changes that either
form an active site of the enzyme or expose
the active site to the substrates.
Wide varieties
• Hormones: proinsulin
• Digestive proteins: trypsinogen, …
• Funtional proteins: factors of blood clotting
and clot dissolution
• Connective tissue proteins: procollagen
Activation of chymotrypsin
1
13 14 15 16
146
149
245
Pro-CT
1
13 14 15 16
146
149
245
1
13
146
149
245
S
16
S
S
S
CT
CT
Features of zymogen activation
• A cascade reaction in general
• To protect the zymogens from being digested
• To exert function in appropriate time and
location
• Store and transport enzymes
§6.1.b Allosteric regulation
• Allosteric enzymes are those whose activity
can be adjusted by reversible, non-covalent
binding of a specific modulator to the
regulatory sites, specific sites on the surface
of enzymes.
• Allosteric enzymes are normally composed
of multiple subunits which can be either
identical or different.
• The multiple subunits are
catalytic subunits
regulatory subunits
• Kinetic plot of v versus [S] is sigmoidal
shape.
• Demonstrating either positive or negative
cooperative effect.
Properties of allosteric enzymes
• There are two conformational forms, T
and R, which are in equilibrium.
• Modulators and substrates can bind to the
R form only; the inhibitors can bind to
the T form.
Allosteric curve
Allosteric activation
Allosteric enzyme
Allosteric represion
[S]
Activation of protein kinase
C
cAMP
R
R
+
C
R
4 cAMP
C
C
+
R
cAMP
cAMP
cAMP
protein kinase
(inactive)
protein kinase
(active)
C: catalytic portions
R: regulatory portions
§6.1.c Covalent modification
• A variety of chemical groups on enzymes
could be modified in a reversible and
covalent manner.
• Such modification can lead to the
changes of the enzymatic activity.
Common modifications
phosphorylation - dephosphorylation
adenylation - deadenylation
methylation - demethylation
uridylation - deuridylation
ribosylation - deribosylation
acetylation - deacetylation
Phosphorylation
ATP
ADP
Mg2+
phosphorylation
protein
kinase
E-O-PO3H2
E-OH
phosphatase
dephosphorylation
Pi
H2O
Features of covalent modification
• Two active forms (high and low)
• Covalent modification
• Energy needed
• Amplification cascade
• Some enzymes can be controlled by
allosteric and covalent modification.
§6.2 Regulation of E Quantity
• Constitutive enzymes (house-keeping):
enzymes whose concentration essentially
remains constant over time
• Adaptive enzymes: enzymes whose quantity
fluctuate as body needs and well-regulated.
• Regulation of enzyme quantity is
accomplished through the control of the genes
expression.
Controlling the synthesis
• Inducer: substrates or structurally related
compounds that can initiate the enzyme
synthesis
• Repressor: compounds that can curtail the
synthesis of enzymes in an anabolic pathway
in response to the excess of an metabolite
• Both are cis elements, trans-acting regulatory
proteins, and specific DNA sequences located
upstream of genes
Controlling the degradation
• Enzymes are immortal, and have a wide range
of lifetime. LDH4 5-6 days, amylase 3-5 hours.
• They degrade once not needed through
proteolytic degradation.
• The degradation speed can be influenced by
the presence of ligands such as substrates,
coenzymes, and metal ions, nutrients and
hormones.
Degradation pathway
• Lysosomic pathway:
–
–
–
–
Under the acidic condition in lysosomes
No ATP required
Indiscriminative digestion
Digesting the invading or long lifetime proteins
• Non-lysosomic pathway:
– Digest the proteins of short lifetime
– Labeling by ubiquitin followed by hydrolysis
– ATP needed
Enzymes/pathways in cellular
organelles
organelle
Enzyme/metabolic pathway
Cytoplasm
Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt,
fatty acids synthesis, purine and pyrimidine catabolism
Mitochondria
Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis,
electron transport chain and oxidative phosphorylation
Nucleus
Biosynthesis of DNA and RNA
Endoplasmic
reticulum
Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid
synthesis and reduction, cytochrome P450, esterase
Lysosomes
Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases
Golgi apparatus
Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyltransferase
Peroxisomes
Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid
oxidase
Section 7
Clinical Applications
§7.1 Fundamental Concepts
• Plasma specific or plasma functional enzymes:
Normally present in the plasma and have
specific functions.
• High activities in plasma than in the tissues.
Synthesized in liver and enter the circulation.
• Impairment in liver function or genetic
disorder leads to a fall in the activities.
• Non-plasma specific or plasma nonfunctional enzymes: either totally absent or at
a low concentration in plasma
• In the normal turnover of cells, intracellular
enzymes are released into blood stream.
• An organ damaged by diseases may elevate
those enzymes
§7.2 Isoenzyme
• A group of enzymes that catalyze the same
reaction but differ from each other in their
structure, substrate affinity, Vmax, and
regulatory properties.
• Due to gene differentiation: the different
gene products or different peptides of the
same gene
• Present in different tissues of the same
system, or subcellular components of the
same cell
Reasons for isoenzyme
• Synthesized from different genes (malate
dehydrogenase in cytosol versus in
mitochondria)
• Oligomeric forms of more than one type of
subunits (lactate dehydrogenase)
• Different carbohydrate content (alkaline
phosphatase)
Lactate dehydrogenase (LDH)
• 5 isoenzymes, LDH1 – LDH5
• Tetramer
– M subunits (M for muscle), basic
– H subunits (H for heart), acidic
• Different catalytic activities
• Used as the marker for disease diagnosis
O
H3C
H
C
OH
Lactate
LDH
COOH
H3C
NAD+
C
NADH + H
Pyruvate
• LDH1 (H4) in heart muscle converts lactate
to pyruvate, and then to acetyl CoA.
• LDH5 (M4) in skeletal muscle converts
pyruvate to lactate.
CO
OH
Creatine phosphokinase
• 3 isoenzymes, BB, BM, MM
• Dimeric form: M (muscle) or B (brain)
• CPK2 is undetectable (<2%) in serum for
healthy individuals, and elevated to 20% in
the first 6-18 hrs after myocardial infarction.
• Used as a earliest reliable indicator of
myocardial infarction.
§7.3 Diagnostic Applications
• Usefulness:
– Enzyme assays provide important information
concerning the presence and severity of
diseases
– Provide a means of monitoring the patient’s
response
• approaches:
– Measuring the enzymatic activities directly
– Used as agents to monitor the presence of
substrates
CM3
LDH1 / LDH2
1.25
CM2
1.00
0.75
1
2
3
Days after myocardial infarction
4
Ratio of LDH1 to LDH2
CK activity
Enzymatic activity changes
Electrophoresis of LDH
Enzymes for disease diagnosis
Serum enzymes
(elevated)
Diseases
Amylase
Acute pancreatitis
Serum glutamate pyruvate
transaminase (SGPT)
Liver diseases (hepatitis)
Serum glutamate oxaloacetate
transaminase (SGOT)
Heart attack (myocardial infarction)
Alkaline phosphatase
Rickets, obstructive jaundice
Acid phosphatase
Cancer of prostate gland
Lactate dehydrogenase (LDH)
Heart attack, liver diseases
γ-glutamyl transpeptidase (GGT)
Alcoholism
5’-nucleotidase
Hepatitis
Aldolase
Muscular dystrophy
§7.4 Therapeutic Applications
• Successful therapeutic uses
– Steptokinase: treating myocardial infarction; preventing
the heart damage once administrated immediately after
heart attack
– Asparaginase: tumor regression
• Several limits
– Can be rapidly inactivated or digested
– May provoke allergic effects
Section 8
Nomenclature
§8.1 Conventional Nomenclature
• Adding the suffix –ase to the name of the
substrates (urease)
• Adding the suffix –ase to a descriptive term
for the reactions they catalyze (glutemate
dehydrogenase)
• For historic names (trypsin, amylase)
• Being named after their genes (Rec A –recA,
HSP70)
§8.2 Systematic Nomenclature
• The International Union of Biochemistry and
Molecular Biology (IUBMB) maintains the
classification scheme.
• Categorize in to 6 classes according to the
general class of organic reactions catalyzed
• Assigned a unique number, a systematic
name, a shorter common name to each
enzyme
§8.2.a Oxidoreductases
1. Catalyzing a variety of oxidationreduction reactions
AH2 + B → A + BH2
2. Alcohol dehydrogenase (alcohol:NAD+
oxidoreductase, E.C. 1.1.1.1.)
Cytochrome oxidase
L- and D-amino acid oxidase
§8.2.b Transferases
1. Catalyzing transfer of a groups between
donors and acceptors
A-X + B → A + B-X
2. Hexokinase (ATP:D-hexose 6phosphotransferase, E.C.2.7.1.1.)
Transaminase
Transmethylases
§8.2.c Hydrolases
1. Catalyzing cleavage of bonds by addition
of water
A-B + H2O → AH + BOH
2. Lipase (triacylglycerol acyl hydrolase, E.C.
3.1.1.3.)
Choline esterase
Acid and alkaline phosphatases
Urease
§8.2.d Lysases
1. Catalyzing lysis of a substrate and
generating a double bond (nonhydrolytic,
and non-oxidative reactions)
A-B + X-Y → AX + BY
2. Aldolase (ketose 1-phosphate aldehyde
lysase, E.C. 4.1.2.7.)
Fumarase
Histidase
§8.2.e Isomerases
1. Catalyzing recemization of optical or
geometric isomers
A → A’
2. Triose phosphate isomerase (Dglyceraaldehyde 3-phosphate
ketoisomerase, E.C. 5.3.1.1.)
Retinol isomerase
Phosphohexose isomerase
§8.2.f Ligases
1. Catalyzing synthetic reactions at the
expense of a high energy bond of ATP
A + B → A-B
2. Glutamine synthetase (L-glutamate
ammonia ligase, E.C. 6.3.1.2.)
Acetyl CoA carboxylase
Auccinate thiokinase
• Blood clot formation and tissue repair are
brought “on-line” only in response to
pressing physiological or
pathophysiological needs.