enzyme lecture ppt.pptx

Intended learning outcomes (ILOs):
By the end of the course, students should be able to:
1. Describe the classification, nomenclature and structure
of enzymes.
2. Identify coenzymes, isoenzymes and cofactors.
3. Understand the mechanism of action and factors
affecting the rate of enzyme action
4. Identify enzyme activators and inhibitors.
5. Correlate enzyme activity with different diseases.

• Enzymes:
- Biological catalysts.
- .
• -
• They accelerate reactions and undergo change during the
reaction but revert to their original state when the reaction is complete.
• - Enzymes increase the reaction rate by lowering the free energy
barrier that separates the reactants and products.
•

The transition state is the transitory of molecular structure in
which the molecule is no longer a substrate but not yet a product
S → X → P, X is the transition state, which is located at the peak
of the curve

Enzyme specificity
• 1-Absolute specificity: Enzymes
only one specific reaction on one
catalyze
specific
molecule e.g. uricase, urease, catalase
•2-Relative Specificity: One enzyme acts on a
group of compound having the same type of
bonds e.g lipase acts on ester bond of
triacylglycerols and phospholipids.

Enzyme classification
• classification of enzymes according to the chemical
reaction type and reaction mechanisms.
• Each enzyme has a code number (EC) that classify
the reaction type to class (first digit), subclass
(second digit) and subsubclass (third digit). The
fourth digit is for the specific enzyme.
EC 2. 7. 1. 1.
Class 2: a transferase.
Subclass 7: transfer phosphate.
Subsubclass 1: the phosphate acceptor is alcohol.
Final digit: denotes the enzyme hexokinase.

The six classes of enzymes include:
(1)Oxidoreductases: Enzymes catalyzing oxidation –
reduction reactions (alcohol dehydrogenase. EC, 1: 1.1.1.)
Alcohol + NAD+ → Aldehyde or ketone + NADH+ H+
(2)Transferases: Enzymes catalyzing transfer of
functional groups (other than H) between two substrates
(choline acyltransferase).
Acetyl- CoA + Choline → CoA + Acetylcholine
(3)Hydrolases: Enzymes catalyzing hydrolysis of a
bond by addition of water (peptidases and proteinases e.g.
trypsin and chymotrypsin).

(4) Lyases:
Enzymes catalyzing group elimination to form
double bonds (fumarase).
L-malate → fumarate + H2O
(5) Isomerases:
Enzymes catalyzing interconversion of isomers by
rearrangement of atomic groupings without altering
molecular weight or number of atoms (aldoses and
ketoses).
(6) Ligases:
Enzymes catalyzing bond formation coupled with
ATP hydrolysis.
ATP + L-glutamate + NH4 → ADP + Pi + L-glutamine.
(glutamine synthase)
ATP + Acetyl CoA + CO2 → ADP + Pi + Malonyl-CoA.
(acetyl-CoA carboxylase)

Cofactors and coenzymes
Some enzymes needs small molecule cofactors to do its action.
These cofactors includes:
1) Metal ions (Cu2+, Fe3+ or Zn2+).
2) Organic molecules (coenzymes) such as :
* Cosubstrate (transiently associated with enzyme).
* Prothetic groups (permanently associated with enzyme.
e.g. heme prothetic group of cytochrome c is tightly bound to
the protein).
Apoenzyme (inactive) + cofactor ↔ holoenzyme (active).
Reaction that require coenzymes include (oxidoreductions,
group transfer, isomerization reactions and ligases).

Types of coenzymes
Coenzymes can be classified to (according
transferred):
1) Coenzymes for transfer groups
hydrogen:
to the group
other than
* Pyridoxal phosphate (B6) for amino group transfer.
* Tetrahydrofolate for one carbon group transfer.
* Thiamin pyrophosphate (B1) for aldehyde transfer.
* Biocytin (from biotin) for carboxylation reactions.

2) Coenzymes for transfer of hydrogen:
* Nicotinamide coenzymes (+) from niacin for
oxidation-reduction reactions. NAD+ and NADP
* Flavin coenzymes (FMN and FAD) from
riboflavin (B2) for oxidation-reduction reactions.
* Coenzyme Q.

APOENZYME and HOLOENZYME
Conjugated protein enzyme (holoenzyme): it consists of
Protein part of the enzyme known as apoenzyme.
Non protein part known as cofactor.

Isoenzymes
Definition:
These are physically distinct forms of a given enzyme
which are present in different cell types (different
tissues).
- They have multiple form but has the same catalytic
activity.
- Differ in their electrophoresis mobility
Isoenzymes may differ in their affinity for substrates.
Examples of isoenzymes: Lactate dehydrogenase
enzyme (LDH), which can be distinguished on the
basis of their electrophoretic mobilities into 5
isoenzymes (LDH1- 5).

LDH isoenzymes
• Each isoenzyme is a tetramer (fromed
from two types of polypeptide, H and M).
H and M subunits are expressed in
different tissues.
• LDH1
(HHMM), LDH4 (HMMM) and
(HHHH), LDH2 (HHHM), LDH3
LDH5
(MMMM).
• LDH1 and LDH2 are predominant in the
heart and red cells (increased in MI),
LDH3 Increase in acute leukemia,
whereas LDH4 and LDH5 are predominant
in the liver and some skeletal muscles.

Functional plasma enzymes:
•. Present in the circulation all time and
perform physiological function in blood.
• Synthesized in the liver.
• Present in blood in equivalent or higher
concentration than tissues.
e.g. lipoprotein lipase, pseudocholinesterase
and proenzymes of blood coagulation
Enzymes important in clinical
diagnosis

(2) Non-Functional plasma enzymes:
• Their presence in the circulation indicates
increased tissue destruction
• perform no physiological function in blood.
• Present in blood in lower concentration than
tissues.
- They include:
• Exocrine enzyme → present in exocrine
secretions and diffuse passively to plasma. e.g.
pancreatic amylase, lipase and alkaline
phosphatase.
• True intracellular enzymes → normally they are
absent from the circulation.

Catalysis acts by decreasing the energy -
barrier between reactants and products, so
making easier to reach the transition state.
Mechanism of action of enzymes

Active site of enzymes
The catalytic (active) site:
Is a restricted region of
the enzyme at which
catalysis takes place.
It is a pocket or groove in
the surface of the protein
(which attract substrate
and mediate catalysis).
There are 2 models of the
catalytic site.

Catalytic site models:
1- Lock & key or rigid template model
- Substrate and enzyme interact like lock and key.
- The substrate binds to a specific site on the enzyme.
- The shape of that site is complementary to that of the
substrate.

2- Induced fit model
The substrate induces a conformational change in the
enzyme, making the enzyme in the correct orientation
for substrate binding, catalysis or both.

Factors affecting enzyme activity:
1 Temperature.
2 pH.
3 Enzyme concentration.
4 Substrate concentration.
5 Inhibitors.

(1) Temperature:
- Over a limited range of T, the velocity of
an enzyme-catalyzed reaction is
increased as the T raises.
- Optimal temperature: It is the T at or
above that of the cells in which the
enzymes occur. At this T the reaction is
rapid, above it the reaction rate
decreases (enzyme denaturation)
(2) pH:
- Moderate pH changes affect the ionic
state of the enzyme and substrate.
- The optimal enzyme activity ranges
between 5-9, except few enzymes (e.g.
pepsin).

(3) Enzyme concentration:
- The velocity of an enzyme-catalyzed reaction is
directly proportional to the enzyme concentration.
- The rate of the reaction is initially constant but gradually
decreases (depletion of substrate, inhibition of the enzyme by
its product or enzyme denaturation).
- So, only the initial velocity (Vi) is used to calculate the
kinetic parameters of the reaction.
- Enzymes change the reaction path but do not affect
the concentrations of the reactants and products.

(4) Substrate concentration:
- The velocity of an enzyme-catalyzed reaction is increased with
increased substrate concentration up to a point where the
enzyme becomes saturated with the substrate.
- Vi (initial velocity) increases and reach Vmax (maximum
velocity) which is not affected by further increase in S
concentration.
K1 K2
Enz + S → EnzS → Enz + P
←
K-1
- Mechaelis-Menten equation describe the relation between Vi,
S, Vmax and rate constants K1, K-1 and K2.
Vmax [S]
Vi = [S] + K-1 + K2
K1

- Km → is the substrate concentration that produces half
maximal velocity (Vmax /2).
Km = K-1 + K2
K1
Vmax [S]
Vi = [S] + Km
Significance of Mechaelis-Menten constant :
When [S] = Km → Vmax [S]
Vi = [S] + [S]
Vi = Vmax
2
* The Km is unique for each enzyme-substrate pair.

Lineweaver-Burk or double reciprocal plot:
- A good method for determining the values of Vmax and Km is
the reciprocal of the Mechaelis-Menten equation.
Vmax [S]
[S] + Km
Vi =
1 [S] + Km
Vi = Vmax [S]
- This is the equation of a straight line. When these quantities are
plotted, we will obtain the Lineweaver-Burk or double reciprocal
plot.

(5) Inhibitors:
Definition:These are substances that reduce enzyme’s
activity, it combines with the enzyme either reversibly or
irreversibly.
(1) Irreversible inhibitiors
 irreversible inhibitors are those :
 that bind covalently with enzyme or
 destroy a functional group on an enzyme => that is essential
for enzyme’s activity.
(2) Reversible inhibitors:
 Competitive
 Uncompetitive
 Non-competitive

A- Competitive inhibitors
- It is a substance that competes directly with a substrate for an
enzyme’s substrate-binding site.
- The chemical structure of the inhibitor (I) resembles that of
the substrate (S).
- High concentration of the S can overcome the effects of the I.
- It increases the Km of the enzyme but has no effect on the Vmax
(In the presence of a competitive inhibitor, it takes a higher
substrate concentration to achieve the same velocities that
were reached in its absence).

Regulation of enzyme activity
(A)Control of enzyme availability:
The amount of a given enzyme
depends on:
- The rate of its synthesis
- The rate of its degradation
↑
in a cell

1- Regulation of enzyme synthesis
Regulation of
enzyme
synthesis
Enzyme
repression
Product
repression
Catabolite
repression
Enzyme
induction
Inducers

2- Regulation of enzyme degradation
Increase enzyme amout by decrease rate
of degradation or decrease in amount of
enzyme by increase the rate of
degradation
- Example
• Liver arginase level is increased in:
- Starvation in animal due to decreased
arginase degradation.
•

B-Control of enzyme activity
1 Allosteric effectors
2 Covalent modification
3 Limited proteolysis

(B) Control of enzyme activity:
1- Allosteric effectors:
- Enzymes bind the effector at an allosteric site that is
physically distinct from catalytic site.
- Allosteric enzymes: are enzymes whose activity at the
catalytic site may be modulated by the presence of allosteric
effectors at the allosteric site.
Feedback inhibition
- Inhibition of the activity of an enzyme in a biosynthetic
pathway by an end product of that pathway.
1 2 3
A → B → C → D
High concentration of D inhibits conversion of A to B. So, D acts
as a negative allosteric effector or feedback inhibitor of the
enzyme, regulating the synthesis of D.

2- Covalent modification:
- The catalytic activity of an enzyme can be
modulated by covalent attachment of a phosphate
group
- These enzymes are termed interconvertable
enzymes.
- The phospho- or dephosphoenzyme may be the
more active catalyst.
- Protein kinases catalyze phosphorylation and
protein phosphatases catalyze dephosphorylation
(hormonal and neural control).

3- Limited proteolysis:
- Enzyme activity can be regulated by converting an
inactive proenzyme to a catalytically active form.
- The proenzyme must undergo limited proteolysis,
which is associated with conformational changes
that create the catalytic site.
- Examples of proenzymes are:
• Digestive enzymes.
• Blood coagulation enzymes.
• Blood clotting enzymes.

enzyme lecture ppt.pptx

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