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Lecture 8
Enzyme
Outline
• Composition, structure and properties
of enzyme
• Enzyme kinetics
• Catalytic mechanisms of enzyme
• Regulation of enzyme activities
1. Introduction to enzymes
(1). Much of the early history of biochemistry
is the history of enzyme research
(2). Biological catalysts were first recognized
in studying animal food digestion and sugar
fermentation with yeast (brewing and wine
making)
(3). Ferments (i.e., enzymes, meaning in “in
yeast”) were thought (wrongly) to be
inseparable from living yeast cells for quite
some time (Louis Pasteur)
(4). Yeast extracts were found to be able to
ferment sugar to alcohol (Eduard Buchner, 1897,
who won the Nobel Prize in Chemistry in 1907
for this discovery)
(5). Enzymes were found to be proteins (1920s
to 1930s, James Sumner on urease and
catalase ,“all enzymes are proteins”, John
Northrop on pepsin and trypsin, both shared the
1946 Nobel Prize in Chemistry)
(6). Catalytic RNA (also called ribozyme --from ribonucleic acid enzyme, or RNA enzyme)
were found in the 1980s (Thomas Cech, Nobel
Prize in Chemistry in 1989)
1. Definition of enzyme
•Enzymes are biological catalysts.
•A Catalyst is defined as "a substance
that increases the rate of a chemical
reaction without being itself changed in
the process.”
What is the difference between an
enzyme and a protein?
Protein
Enzymes
RNA
•All enzymes are proteins except some RNAs
• not all proteins are enzymes
Enzymes are the most remarkable
and specialized biological
catalysts
• An enzyme catalyzes a chemical reaction at a
specifically structured active site, being often a pocket.
• Enzymes have extraordinary catalytic power, often far
greater than those non-biological catalysts.
• Enzymes often have a high degree of specificity for
their substrates.
• Enzymes are often regulatory.
• Enzymes usually work under very mild conditions of
temperature and pH.
• The substance acted on by an enzyme is called a
substrate, which binds to the active site of an enzyme
in a complementary manner.
2. How enzymes work (important!)
1) Enzymes lower a
reaction’s activation
energy
– All chemical reactions
have an energy barrier,
called the activation
energy, separating the
reactants and the
products.
– activation energy:
amount of energy
needed to disrupt
stable molecule so that
reaction can take place.
Enzymes
Lower a
Reaction’s
Activation
Energy
2) The active site of the enzyme
• Enzymes bind substrates to their active
site and stabilize the transition state of
the reaction.
• The active site of the enzyme is the place
where the substrate binds and at which
catalysis occurs.
• The active site binds the substrate,
forming an enzyme-substrate(ES) complex.
Binding site
Active site
Catalytic site
Enzymatic reaction steps
1.
2.
3.
4.
5.
Substrate approaches active site
Enzyme-substrate complex forms
Substrate transformed into products
Products released
Enzyme recycled
Characteristics of active sites
• The active site takes up a small part
of the total volume of the enzyme.
• The active site is 3-dimensional and
is generally found in a crevice or
cleft on the enzyme.
• The active site displays highly
specific substrate binding.
Active center of lysozyme
129 aa, discovered by Fleming in 1922 in tears
Active center may include distant residues
3. Properties of enzymes
(important!)
• Catalytic efficiency – high efficiency, 103 to
1017 faster than the corresponding
uncatalyzed reactions
• Specificity - high specificity, interacting with
one or a few specific substrates and
catalyzing only one type of chemical reaction.
• Mild reaction conditions- 37℃, physiological
pH, ambient atmospheric pressure
High specificity
1). Absolute specificity: the enzyme will
catalyze only one reaction.
e.g
NH 2
C O
NH 2
H2O
脲酶
Urease
NH 2
C O
NHCH 3
H2O
脲酶
Urease
CO2 + 2NH3
X
2). Relative specificity
(i) Group specificity: the enzyme will act only
on molecules that have specific functional
groups, such as amino, phosphate and methyl
groups.
A—B
e.g
or
A—B
α-D-glucosidase
CH 2OH
O
OH
O
OH
OH
R
(ii) Bond specificity: the enzyme will act
on a particular type of chemical bond
regardless of the rest of the molecular
structure.
O
esterase
酯酶
+ H2O
R1C
R1COOH + R2OH
OR2
3). Stereospecificity: the enzyme will act
on a particular steric or optical isomer.
H C COOH
HOOC C H
fumarate
hydratase
延胡索酸水化酶
+ H2O
CH2COOH
CHOHCOOH
malate
4). Hypotheses of enzyme
specificity
(i) Lock and Key model
Proposed by Fischer in 1894
In this model, the active sites of the unbound
enzyme is complementary in shape to the
substrate
(ii) Induced-fit model
Proposed by Koshland in 1958
In this model, the enzyme changes shape
on substrate binding
An Example: Induced conformational
change in hexokinase
•Catalyzes phosphorylation of glucose to glucose 6phosphate during glycolysis
•such a large change in a protein’s conformation is not
unusual
Conclusion
• Enzymes lower the free energy of
activation by binding the transition state of
the reaction better than the substrate.
•
The enzyme must bind the substrate in
the correct orientation otherwise there
would be no reaction.
•
Not a lock & key but induced fit – the
enzyme and/or the substrate distort
towards the transition state.
4 Chemical composition of enzymes
(1) Simple protein
(2) Conjugated protein
Holoenzyme= Apoenzyme+ Cofactor
Cofactor
Coenzyme : loosely bound to enzyme (noncovalently bound).
Prosthetic group : very tightly or even
covalently bound to enzyme (covalently bound)
• Cofactors often function as transient carriers
of specific (functional) groups during catalysis.
• Many vitamins and organic nutrients required
in small amounts in the diet, are precursors of
cofactors.
5 Classification of enzymes
(1). By their composition
1). Monomeric enzyme
2). Oligomeric enzyme
3). Multienzyme complex: such as
Fatty acid synthase
(2) Nomenclature
• Recommended name
•Enzymes are usually named according to the
reaction they carry out.
•To generate the name of an enzyme, the
suffix -ase is added to the name of its
substrate (e.g., lactase is the enzyme
that cleaves lactose) or the type of
reaction (e.g., DNA polymerase forms
DNA polymers).
•Systematic name (International classification)
• By the reactions they catalyze (Six classes)
Lactate
dehydrogenase
NMP kinase
Chymotrypsin
Fumarase
Transfer electrons (hydride ions or H a
play a major role in energy metabolism.
e.g., the transfer of a phosphoryl group
from ATP to many different acceptors.
i.e., the transfer of functional groups to
These are direct bond breaking reactions
without being attacked by another reactant
such as H2O.
Triose phosphate
isomerase
Leading to the formation of C-C, C-S,
C-O, C-N bonds.
Aminoacyl-tRNA synthetase
Each enzyme is given a systematic name and a unique 4digit identification number for identification by the
Enzyme Commission (E.C.) of IUBMB (since 1964)
lactate + NAD+
pyruvate + NADH + H+
Lactate dehydrogenase (lactate:NAD+ oxidoreductase)
1
Indicates
type of
substrate
Indicates
type of
cofactor
6 Catalytic mechanisms of enzymes
• Mechanisms - the molecular details of
catalyzed reactions
• How do enzymes stabilize the
transition state of a reaction
–
–
–
–
General Acid-base catalysis
Covalent catalysis
Catalysis by proximity and orientation
Metal catalysis
1). General acid-base catalysis
• The active sites of some enzymes contain
amino acid functional groups that can
participate in the catalytic process as proton
donors or proton acceptors --general acidbase catalysis.
• A general acid (BH+) can donate protons
• A covalent bond may break more easily if
one of its atoms is protonated
Amino acids in general acid-base catalysis
2). Covalent catalysis
• Covalent catalysis involves the substrate
forming a transient covalent bond with
residues in the active site of the enzyme or
with a cofactor.
• This adds an additional covalent intermediate
to the reaction, and helps to reduce the
energy of later transition states of the
reaction.
• Group X can be transferred from A-X to B in two
steps via the covalent ES complex X-E
A-X + E
X-E + A
X-E + B
B-X + E
nucleophilic
center (X:)
•Examples of covalent bond formation between
enzyme and substrate.
•In each case, a nucleophilic center (X:) on an
enzyme attacks an electrophilic center on a
substrate.
Nucleophilic
group (X:)
Electrophilic
group
3). Catalysis by proximity and orientation
• This increases the
rate of the
reaction as
enzyme-substrate
interactions align
reactive chemical
groups and hold
them close together.
•Analogous to an
effective
increase in
concentration of
the reactants.
4).Many enzymes have metal
ions in their active centers
playing important roles in
catalysis.
– help activate substrates
– Stabilize charged transition
states by forming ionic bonds.
Substrate
Enzyme
•An enzyme may use a combination of
several catalytic strategies to bring about a
rate enhancement.
7. Enzyme activity
• Enzymes are never expressed in terms
of their concentration (as mg or μg
etc.), but are expressed only as
activities.
• Enzyme activity = moles of substrate
converted to product per unit time.
– The rate of appearance of product or the
rate of disappearance of substrate
– Test the absorbance: spectrophotometer
Units of enzyme activity:
•
Katal (kat) – 1 kat denotes the
conversion of 1 mole substrate per
second (mol/sec)
•
International unit (IU) - amount of
enzyme activity that catalyses the
conversion of 1 micromol of substrate
per minute (μmol/min).
8. Factors affecting enzyme activity
•
•
•
•
•
•
Concentration of substrate
Concentration of enzyme
Temperature
pH
Activators
Inhibitors
Enzyme velocity
• Enzyme activity is commonly expressed
by the intial rate (V0) of the reaction
being catalyzed. (why?)
• Enzyme activity =
moles of substrate
converted to product
per unit time.
• Velocity decreases as time increases as:
– S may be used up
– P may inhibit reaction(E)
– Change of pH may occur and decrease
enzyme reaction
– Cofactor or coenzyme may be used up
– Enzyme may loss activity
(1). Substrate concentration ([S]) affects
the catalytic velocity (rate)
C
B
A
•At relatively low concentrations
of substrate, Vo increases
almost linearly (A) with an
increase in [S].
•At higher [S], Vo increases by
smaller and smaller amounts (B)
in response to the increase in
[S].
• Finally, a point (a plateau of
maximum velocity, Vmax) (C) is
reached.
•[S] is a key factor
affecting the rate.
Quantitative expression of
relationship between [S] and V0
•1913, Leonor Michaelis and Maud Menten
deduced the equation, Michaelis-Menten equation,
based on the exist of intermediate ([ES]) in the
enzyme reaction.
Leonor Michaelis
(1875-1949)
Maud Menten
(1879-1960)
Proposed Model
• ES complex formed when specific substrates
fit into the enzyme active site (at the
beginning of reaction)
E + S
k1
k2
ES
k3
E + P
• When [S] >> [E], E is saturated with S.
• k1,k2 and k3 represent the velocity constants
for the respective reactions.
Michaelis-Menten equation (very important!)
• Michaelis-Menten equation describes how
reaction velocity (V) varies with substrate
concentration [S].
• The following equation is obtained after
suitable algebraic manipulation.
V = Vmax
[S]
[S] + KM
Note: V means V0
Km: Michaelis constant
Km = (k2 + k3)/k1
[S]
V
=
Vmax
[S] + Km
 The equation fits the
observed curve very well.
•When [S] is very low (<<Km),
then V = (Vmax/ Km)[S] or V
Zero order
is linearly dependent on [S].
reaction
First order
reaction
•when [S] is very high (>>Km),
then V = Vmax; that is, the
V is independent of [S].
Significances of Km
1)
When [S] = Km,
Vmax [S]
Vmax [S]
Vmax
V =
=
=
Km+[S]
[S] + [S]
2
so, when V = 1/2 Vmax , Km = [S], the unit of Km is as [S]
2) For a specific substrate, Km is a constant for the
enzyme.
3) Km can be a measure of the affinity of E for S. A low
Km value indicates a strong affinity between E and S.
• The lower the value of Km, the stronger affinity
between E and S
Significances of Vmax
When [S] is very much greater than Km,
Vmax [S]
Vmax [S]
V=
=
= Vmax
Km+[S]
[S]
• Vmax is a constant.
• Vmax is the theoretical maximal rate of
the reaction - but it is NEVER achieved
in reality.
Measurement of Km and Vmax
(i) Plot of V vs [S]
If [S]<<Km
If [S]=Km
If [S]>>Km
•Using the MichaelisMenten equation, the
Vmax is an asymptote
and can thus only be
approximated and as a
result, the Km, which
is Vmax/2, can't be
determined accurately.
(ii) The double-reciprocal
--Lineweaver-Burk plot
Transform the Michaelis-Menten equation to
1 Km 1
1



v Vmax [S] Vmax
The double-reciprocal
Lineweaver-Burk plot is
a linear transformation
of the Michaelis-Menten
plot (1/V vs 1/[S])
(y=ax+b)
(2) Effect of [E] on velocity
[S]>>[E]
V∝[E]
• The initial rate of an
enzyme-catalyzed
reaction is always
proportionate to the
concentration of enzyme.
• This property of enzyme
is made use in
determining the serum
enzyme for the diagnosis
of diseases.
(3) Effect of temperature on
velocity Bell-shaped curve
•It is worth noting that the
enzymes have been assigned
optimal temperatures based on
the laboratory work.
(4) Effect of pH value on velocity
Bell-shaped curve
•The pH optimum varies for
different enzymes.
•Most enzyme: neutral pH (6-8).
• Each enzyme has
an optimal pH or pH
range (where the
enzyme has maximal
activity).
• Requirements for
the catalytic groups
in the active site in
appropriate
ionization state is a
common reason for
this phenomenon.
(5) Effect of activator on velocity
•Enzyme activators are molecules that bind to
enzymes and increase their activity.
(i). Inorganic ions
• Metal ions，such as Na+, K+, Mg2+, Ca2+, Cu2+,
Zn2+, Fe2+ et al
• Anions: such as Cl-, Br-, I-、CN- et al
(ii).
Organic
• Reducing agents, such as Cys、GSH
(iii).
Proteins
(6)
Inhibition of enzyme activities
(very important!)
• Inhibitor: any molecule which acts
directly on an enzyme to lower its
catalytic rate is called an
inhibitor.(not denaturation)
• Some enzyme inhibitors are normal
body metabolites.
• Other may be foreign
substances,such as drugs or toxins.
Significance of studying inhibition of enzy
• Relationship of structure & function of enzyme
• Mechanisms of enzyme catalysis
• Design of new drugs
Inhibition
• Irreversible inhibition
• Reversible inhibition
– competitive inhibition
– non-competitive inhibition
– uncompetitive inhibition
1). Irreversible
inhibition
• An irreversible
Enzyme
O
I
S
inhibitor
binds tightly, often
covalently, to amino acid
residues at the active
site of the enzyme,
inactivating the enzyme.
•Irreversible inhibition is different from irreversible
enzyme inactivation.
•Irreversible inhibitors are generally specific for one
class of enzyme and do not inactivate all proteins; they
do not function by destroying protein structure but by
specifically altering the active site of their target.
Examples: diisopropyl fluorophosphate( DFP)
• DFP is an organic phosphate that inactivates serine
proteases, it can react with the active site serine
(Ser-195) of enzyme to form DFP-E.
• These inhibitors are toxic because they inhibit
acetylcholin esterase (a serine protease that hydrolyzes
the neurotransmitter acetylcholine).
• Such organophosphorous inhibitors are used as
insecticides or for enzyme research.
DFP
Heavy metals
• Many poisons are harmful to cells because they
are potent irreversible inhibitors. Examples are
heavy metals (mercury, lead, silver, ……)
• Heavy metals such as Ag+, Hg2+, Pb2+ have
strong affinities for sulfhydral (-SH) groups.
• Since many enzymes contain -SH as part of
their active sites, any chemical which can react
with them acts as an irreversible inhibitor.
Enzyme
Enzyme
2). Reversible enzyme inhibition
• Inhibitor ( I ) binds to an enzyme and prevents
formation of ES complex or breakdown to E +
P.
• Reversible inhibitors bind to the enzyme via
noncovalent interactions and can be dissociated
readily from the enzyme.
• There are three basic types of inhibition:
Competitive, Noncompetitive and Uncompetitive.
(i) Competitive inhibition
• Competitive inhibitors usually
resemble the substrate
• Substrate cannot bind when I is
bound at active site
(S and I “compete” for the
enzyme active site)
• Inhibitor
binds only to
free enzyme
(E) not (ES)
• The effect of a competitive
inhibitor can be overcomed
with high concentrations of the
substrate
Example 1 - competitive inhibition
• Malonate is a
competitive
inhibitor of
succinate for
succinate
dehydrogenase
Clinical significance of enzyme
inhibition
• The usefulness of the most important
pharmaceutical agents, antimetabolites, is
based on the concept of competitive enzyme
inhibition.
• The antimetabolites are structural analogues
of normal biochemical compounds.
• As competitive inhibitors they compete with
the naturally substrate for the active site of
enzyme and block the formation of
undesirable metabolic products in the body.
• They are in use for cancer therapy, gout etc.
Example - Competitive Inhibition
NH2
folic acid
COOH
p-aminobenzoic acid
NH2
SO2 NH2
sulfanilamide
• Sulfanilamides (also
known as sulfa
drugs, discovered in
1932) were the first
effective systemic
antibacterial agents.
• Sulfanilamide is a
competitive inhibitor
of p-aminobenzoic
acid (PABA).
• p-aminobenzoic acid (PABA) is required by many
bacteria to produce an important enzyme cofactor,
THF. These bacteria require THF for their growth
and division.
•Sulfanilamide acts
as a competitive
inhibitor to enzymes
that convert PABA
into folic acid,
resulting in a
depletion of this
cofactor.
•The depletion of
this cofactor results
in retarded growth
and eventual death
of the bacteria.
• Mammals absorb their folic acid from
their diets, so sulfanilamide exerts no
effects on them.
Bacteria: PABA
DHF
THF
Mammal: Folic acid
(diet)
DHF
THF
Kinetics of competitive inhibition
Direct and Lineweaver-Burk plot
Vmax
vo
I
Km Km’
[S], mM
•Vmax does not change
•At a sufficiently high [S], the reaction velocity reaches the
Vmax observed in the absence of inhibitor.
•Km increases
•This means that in the presence of a competitive inhibitor
more substrate is needed to achive ½ Vmax.
(ii) noncompetitive inhibition
• The inhibitor binds at a
site other than the active
site on the enzyme surface.
• This binding impairs the
enzyme function.
• The inhibitor has no
structural resemblance with
the substrate.
• Inhibition cannot be
overcome by addition of S.
noncompetitive inhibition
Inhibitors bind to both E and ES
Kinetics of noncompetitive inhibition
Direct and Lineweaver-Burk plot
Vmax
vo
Vmax’
I
Km
= Km’
[S], mM
•Vmax decreases
•Noncompetitive inhibition cannot be overcome by increasing [S].
•Km does not change
•Noncompetitive inhibitors do not interfere with the binding of
substrate to enzyme.
(iii) Uncompetitive inhibition
•Inhibitors bind to ES not to free E.
•The effect of an uncompetitive
inhibitor can not be overcome by high
concentrations of the substrate.
Kinetics of uncompetitive inhibition
Direct and Lineweaver-Burk plot
V0
Vmax
I
Km’
Km
Vmax’
[S], mM
Vmax decreased
Km also decreased
•Lines on doublereciprocal plots are
parallel.
•This type of inhibition
usually only occurs in
multisubstrate reactions.
Enzyme Inhibition (summary)
I
Competitive I Non-competitive
Equation and Description
Cartoon Guide
Substrate
E
S
S
E
I
Compete for
Inhibitor active site
E + S←
→ ES → E + P
+
I
↓↑
EI
S
I
I
Uncompetitive
S
E
I
I
Different site
E + S←
→ ES → E + P
+
+
I
I
↓↑
↓↑
EI + S →EIS
[I] binds to free [E] only, [I] binds to free [E] or [ES]
and competes with [S];
complex; Increasing [S] can
increasing [S] overcomes
not overcome [I] inhibition.
Inhibition by [I].
S
I
E + S←
→ ES → E + P
+
I
↓↑
EIS
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
Enzyme Inhibition (Plots)
I
Competitive
Non-competitive
I
Direct Plots
Vmax
vo
vo
I
Double Reciprocal
Km Km’
I
[S], mM
Km = Km’
Uncompetitive
I
Vmax
Vmax
Vmax’
Vmax’
[S], mM
I
Km’
Km
[S], mM
Vmax Unchanged
Km Increased
Vmax Decreased
Km Unchanged
Both Vmax & Km Decreased
1/vo
1/vo
1/vo
Intersect
at Y axis
1/Km
I
I
I
Two parallel
lines
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
1/ Vmax
1/[S]
1/ Vmax
1/Km
1/[S]
9. Regulation of enzyme
activities
• The activity of some enzymes are precisely
and tightly regulated in living organisms to
meet physiological requirements.
• Regulatory enzymes - activity can be
reversibly modulated by effectors.
• Such enzymes are usually found at the first
unique step in a metabolic pathway.
• Regulation at this step conserves material
and energy and prevents accumulation of
intermediates.
Regulation of enzyme activity
• Allosteric enzyme
• Reversible covalent modification
• Proteolytic activation
(1).
Allosteric
regulation
• Allosteric enzymes have a second regulatory
site (allosteric site, Greek: allo-other)
distinct from the active site.
• Allosteric inhibitors or activators bind to
this site (noncovalently) and regulate enzyme
activity via conformational changes.
• Regulatory enzymes possess quaternary
structure (contain multiple subunits).
• There is a rapid transition between the
active (R) and inactive (T) conformations
R
T
• There is a rapid transition between the active (R) and
inactive (T) conformations.
• Substrates and activators may bind only to the R
state while inhibitors may bind only to the T state.
sigmoidal-shaped curve
• A plot of V0 against [S] for an allosteric
enzyme gives a sigmoidal-shaped curve
(normal enzyme is a hyperbolic curve).
(2). Reversible covalent
modification
1). What’s covalently modulated enzymes?
•Activity is modulated by covalent
modification of one or more of its amino
acid residues in the enzyme molecule.
• Common modifying groups include:
phosphoryl, adenylyl, methyl, hydroxyl,
sulfate and ADP-ribosyl groups.
• These groups are generally linked to
and removed from the regulatory
enzyme by separate enzymes.
Phosphorylation and dephosphorylation
• The most common such modification is the
addition and removal of a phosphate group:
phosphorylation and dephosphorylation,
respectively.
• Phosphorylation is catalyzed by protein
kinases, often using ATP as the phosphate
donor.
• Dephosphorylation is catalyzed by protein
phosphatases.
Protein kinases catalyze the
phosphorylation of proteins
Protein phosphatases remove phosphate
groups from phosphorylated proteins
ATP Protein Kinase ADP
OH
+
+
Pi Protein Phosphatase
•Phosphorylation and
dephosphorylation are
not the reverse of one
another.
O • The rate of cycling
OP O between the
O-
phosphorylated and
the dephosphorylated
states depends on the
relative activities of
kinases and
phosphatases.
Response of enzyme to phosphorylation
• Depending on the specific enzyme,the
phosphorylated form may be more or less
active than the unphosphorylated enzyme.
• For example,phosphorylation of glycogen
phosphorylase (GP,an enzyme that degrades
glycogen) increases activity, whereas the
addition of phosphate to glycogen synthase
(GS,an enzyme that synthesizes glycogen)
decrease activity.
(active form)
(inactive form)
(inactive form)
(active form)
Phosphorylation Is a Highly Effective Means of
Regulating the Activities of Target Proteins
Inactive protein
kinase 1
Inactive protein
kinase 2
Inactive protein
kinase 3
Inactive protein
kinase 4
Inactive protein
kinase 1
Inactive protein
kinase 5
Phosphatase
Phoshporylation cascade
Active protein
Cellular response
(3). Zymogen or proenzyme
• Some enzymes are synthesized as larger
inactive precursors calles proenzymes or
zymogens.
• These are activated by the irreversible
hydrolysis of one or more peptide bonds.
• The pancreatic proteases trypsin,
chymotrypsin and elastase are all derived
from zymogen precursors by proteolytic
activation.
autocatalysis
in the small intestine
cascade
The central role of trypsin: it
is the common activator of all
pancreatic zymogens.
Activation of chymotrypsinogen to chymotrypsin,
and of trypsinogen to trypsin
Acute pancreatitis
• Premature activation of these zymogens
leads to the condition of acute pancreatitis.
– The exocrine pancreas produces a variety of
enzymes, such as proteases, lipases, and
saccharidases.
– These enzymes contribute to food digestion.
– In acute pancreatitis, the worst offender among
these enzymes may well be the protease
trypsinogen which converts to the active trypsin
which is most responsible for auto-digestion of
the pancreas which causes the pain and
complications of pancreatitis.
10. Isoenzyme or isozyme
Definition:
• Enzymes in an organism that catalyze the
same reaction but differ in structure;
these differences may range from one to
several amino acid residues.
Lactate dehydrogenase (LDH)
H
Heart type
HH
HH
HH
HM
M
Muscle type
MM
MM
HH
MM
HM
MM
• LDH is a tetramer of two
different types of subunits,called
H and M, which have small
differences in amino acid sequence.
•The two subunits can combine
randomly with each other,forming
5 isoenzymes that have the
compositions H4, H3M, H2M2, HM3,
M4.
11. Enzymes in clinical diagnosis
• An enzyme test is a blood test or
urine test that measures levels of
certain enzymes to assess how well
the body’s systems are functioning
and whether there has been any
tissue damage. (why?)
Plasma enzymes
• Plasma enzymes can be classified into two
major groups:
1) a relatively small group of enzymes are actively
secreted into the plasma by certain organs.
• For example,the enzymes involved in blood coagulation.
2) a large number of enzyme species are released
from cells during normal cell turnover.These
enzymes are normally intracellular and have no
physiologic function in the plasma.
• In healthy individuals, the levels of these enzymes are
fairly constant.
• The presence of elevated enzyme activity in the
plasma may indicate tissue damage.
• Common enzymes used for clinical
diagnosis include:
– alanine aminotransferase(ALT,also called
glutamate pyruvate transaminase,GPT)
– alkaline phosphatase
– amylase
– aspartate aminotransferase
– creatine kinase
– lactate dehydrogenase
Points
• How enzymes work
– lower a reaction’s activation energy, active site
• Properties of enzymes
– highly efficiency, highly specific, mild reaction
conditions,
– Hypothese of enzyme specificity: induced-fit model
• Classification of enzymes: six classes
• Enzyme kinetics
– Substrate Concentration: Michaelis-Menten equation,
Km, Vmax, double-reciprocal plot
– Inhibition of enzyme activities: Irreversible inhibition,
Reversible inhibition (competitive, non-competitive,
uncompetitive inhibition)
• Regulation of enzyme activities
– Allosteric enzyme, Reversible covalent modification,
Proteolytic activation
• Isoenzyme