Download Unit-III Enzymes

Unit-III
Enzymes
Contents
1. Introduction and Properties of enzymes
2. Nomenclature and Classification
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
Introduction and
Properties of Enzymes
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.
Catalyzed reactions
• Reactants need to pass over the energy
barrier, ΔG+.
• Catalysts reduce the activation energy
and assist the reactants to pass over the
activation energy.
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
Characteristics
Enzyme-catalyzed reactions have the following characteristics in
comparison with the general catalyzed reactions:
•
common features: 2 “do” and 2 “don’t”
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: 3 “high”
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.
Classification
Oxidoreductases
Transferases
Hydrolases
Lysases
Isomerases
Ligases
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)
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 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
+ H2 O
C
NH2
urea
NH CH3
O
C
+ H2 O
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.
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
H3 C
A
B
C
Lactate dehydrogenase can recognize only the L-form
but the D-form lactate.
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.
Components of Enzymes
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
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.
Mechanism of EnzymeCatalyzed Reactions
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.
Induced structural changes
Kinetics of EnzymeCatalyzed Reactions
Intermediate state
Forming an enzyme-substrate complex,
a transition state, is a key step in the
catalytic reaction.
k1
ES
E + S
k3
E + P
k2
initial
intermediate
final
Rate constants
k1
ES
E + S
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
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. Km
k2 + k3
=
k1
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].
Factors affecting enzyme-catalyzed
reaction
• Substrate concentration
• Enzyme concentration
• Temperature
• pH
• Inhibitors
• Activators
Inhibition of Enzyme
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.
Summary of inhibition
type
binding target
Km
Vmax
Competitive
E only
⇑
=
Noncompetitive
E or ES
=
⇓
Uncompetitive
ES only
⇓
⇓
Activators
• Metal ions
• essential activators: no enzymatic
activity without it
Mg2+ of hexokinase
• non-essential activators: enhancing the
catalytic power.
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.
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.
Regulation of Enzyme Activity
• Zymogen activation
• Allosteric regulation
• Covalent modification
Clinical Applications
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 non‐
functional 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
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
•
(e.g) Lactate dehydrogenase (LDH)
•
Creatine phosphokinase
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
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
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