Enzymes

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 Thousands of chemical reaction reactions proceeding
very rapidly at any given time within living cells

 Transformation are catalyzed by enzymes which are

usually protein in nature.

 Enzymes are biocatalysts accelerating the rate of

chemical reactions.

 Enzyme catalysis is very rapid, one molecule of

enzyme can act on 1000moles of substrate/min.
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 Lack of enzymes lead to block in the metabolic
pathways causing a group of diseases termed inborn
errors of metabolism.

Definitions and terminology

 Apoenzyme; is the protein part of the enzyme.

 Coenzyme; is a nonprotein, low Mwt, heat stable substance

binding loosely with the enzyme and regenerated after the
reaction (few coenzyme can bind firmly “covalently” and they
are termed prosthetic group).

 Holoenzyme: represents the enzyme and its coenzyme.

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 Substrate: the molecule upon which the enzyme act to
form the product.

 Substrate binding site (active site): particular region

on the surface having a specific arrangement of
chemical groups formulated to bind a specific substrate.

 Allosteric sites: some enzymes contain other sites

“allosteric sites” where small molecules (allosteric
effectors) can bind resulting in increased or decreased
activity of the enzyme for its substrate.
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Intracellular Location of
enzymes :
presented the metabolic pathway
in each cell compartment.
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Nomenclature
 Classification of enzymes
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1. Oxidoreductases: catalyze oxidation and reduction reactions. Use
oxygen as an electron acceptor but do not incorporate it into the
substrate.

Examples:
Dehydrogenases: Use molecules other than oxygen (e.g, NAD+ ) as an
electron acceptor.

Oxygenases: directly incorporate oxygen into the substrate.

Peroxidases: use H2O2 (hydrogen peroxide) as an electron acceptor.

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2. Transferase: transfer groups other than O2 and H.

Methyltransferases: transfer methyl groups between substrates.

Aminotransferases: transfer NH2 from amino acids to keto acids,

Kinases: transfer PO3− from ATP to substrate, e.g., Hexokinase:

Hexokinase

Phosphorylases: transfer PO3− from inorganic phosphate to substrate.

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3.Hydrolases: Hydrolyse in the presence of water.

Phosphatases: remove PO3− from substrate.

Phosphodiesterases: cleave phosphodiester bonds such as those in
nucleic acid.
Proteases: Cleave amide bonds such as those in proteins.
4. Lyases: cleaves C-C , C-S or certain C-N bonds without addition of
water. Some call it synthase (form new product without using ATP).
Decarboxylases: produce CO2 via elimination reaction.
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5.Isomerase: interconvert isomers, for example:
Racemases: interconvert L (levorotatary) and D (dextrorotatary)
stereoisomers.
Mutases: transfer groups between atoms.

6.Ligases: catalyze formation of bonds between C and O, S, N

coupled to hydrolysis of high energy phosphate (ATP).
Carboxylase: add CO2 to substrate.

Synthetases: link 2molecules via an ATP-dependent reaction.

 19 Catalytic activity

Substrate specificity and the active site:

An enzyme catalyzed reaction is initiated when the
enzyme binds to form an enzyme-substrate complex .

In general enzyme molecules are larger than substrate

molecules.

Binding occurs at the active site of the enzyme.

The unique catalytic properties of the enzyme are based

on its 3-dimensional structure and on the active site
whose chemical groups may be brought into close
proximity from different regions of the polypeptide chain.
 20 Active site of enzyme
1. Active site occupies a small part of the enzyme and is situated in a cleft
in the enzyme where the substrate binds.

2. Binding of the substrate to the active site depends on presence of

specific groups or atoms in the active site for the substrate binding and
catalysis.

3. During binding, these specific groups may realign themselves to

provide the unique conformation permitting exact fitting of the
substrate in the active site.

4. Binding of substrate to the active site is through non-covalent bonds

(electrostatic bonds, hydrogen bonds and hydrophobic interactions).

5. Amino acid residues at the active site are called catalytic residues and
catalysis occurs at this site.
20 Models are postulated to explain substrate binding to the enzyme:
First Model:
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Second Model: Induced Fit Model:
The binding site is not fully formed. Binding of the substrate to the
enzyme will induce a conformational change in the enzyme directing
appropriate amino acids to the active site. Some times, these changes
are accompanied by changes in the substrate to provide a perfect fit
for substrate binding and catalysis.

Induced Fit Model

 23 Specificity of Enzymes
Enzymes are highly specific and catalyze only one type of
reaction.
1. Absolute specificity: The enzyme is specific for one
substrate e.g.; urease acts only on urea; glucose oxidase
oxidizes only glucose but not other monosaccharides.
2. Relative specificity: The enzyme acts on a group of closely
related substrates: pancreatic lipase hydrolyzes alpha ester
bonds in triglycerides irrespective of the nature of fatty acid
attached. (bond specificity).
3. Group specificity: Most proteolytic enzymes show group
specificity, for example; trypsin hydrolyzes peptide bonds
provided only by arginine and lysine. (bond and group
specificity).
4. Stereospecificity: Human enzymes are specific for L-amino
acids and D-monosaccharides.
24 Activation Energy

1 Imagine a chemical reaction

as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products).

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 Activation Energy

1 Imagine a chemical reaction

as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products).

2 Activation energy is the

energy needed to roll the stone
up the hill.

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 Activation Energy

1 Imagine a chemical reaction

as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products). 3 Once over the hill, the rest of
the reaction occurs.

2 Activation energy is the

energy needed to roll the stone
up the hill.

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 Activation Energy

1 Imagine a chemical reaction

as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products). 3 Once over the hill, the rest of
the reaction occurs.

2 Activation energy is the

energy needed to roll the stone
up the hill. 4 The stone rolls down and breaks into
tiny pieces (products are formed).

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 Activation Energy

1 Imagine a chemical reaction

as the process of rolling a huge
stone (reactant) up a hill so
that it rolls down and breaks
into tiny pieces (products). 3 Once over the hill, the rest of
the reaction occurs.

2 Activation energy is the

energy needed to roll the stone
up the hill. 4 The stone rolls down and breaks into
tiny pieces (products are formed).

5 The energy needed to start a chemical

reaction is called activation energy.
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25 Enzymes as catalysts Enzymes lower the activation
energy of a reaction so that it occurs more readily.

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Factors affecting enzyme activity

1) Temperature.
2) pH
3) Substrate concentration.
4) Enzyme concentration.
5) Coenzymes and cofactors.
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Most enzymes pH 6-8

Blood pH 7.4

Pepsin pH 2
Acid phosphatase 4-5
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 32 Features of coenzymes
i. Coenzymes are heat stable and mostly derived from vitamins.

ii. They are low molecular weight substances.

iii. The coenzyme combines loosely to the enzyme by non-covalent

linkage. When the reaction completed, the coenzyme is released
from the apoenzyme.

Coenzymes derived from water-soluble vitamins (B-complex group)

can be divided into 2 groups:

a) Coenzymes involved in hydrogen transfer reactions: they donate

or accept hydrogen or electrons, e.g., NAD+, NADP+ , FAD+ and.
Lactate + NAD+ Pyruvate + NAD+ + H +
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b) Coenzymes taking part in reactions transferring a group other than
H+:
CO2 Biotin.
NH2 Pyridoxal phosphate (PLP).

Effect of Metals:
 Metal cofactors may bound reversibly or tightly to enzymes.

 Reversible binding occurs in Metal activated enzymes (e.g.

magnesium in kinases and phosphotransferase).

 Tight binding occurs in metalloenzymes (e.g. Ca2+ for lipases).

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 37 Effect of substrate concentration
The initial rate ( or initial velocity, Vi) of an enzyme catalyzed reaction
is dependent on substrate concentration [ S ]. If substrate concentration
[ S ] increased, while other conditions kept constant, the initial
velocity, Vi “point A” (velocity measured when very little substrate has

Velocity (V)

Vmax
C
Vmax /2
Vmax /2
B
Vmax /2
Vi A

Km
Substrate conc.[ S ]
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Reacted) increased to a maximum velocityVmax (point C) with no
subsequent increase.
Plotting the velocity of an enzyme catalyzed reaction at different
substrate concentration is demonstrated in the figure:
 Point A represents the initial velocity; small number of enzymes is
occupied with the substrate [ES].
 Point C represent maximal velocity (Vmax ); all free enzymes are
saturated with the substrate and present as [ES].
 Point B half of the enzyme molecules are saturated with the
substrate, velocity is half maximal velocity (Vmax /2) at this enzyme
concentration. The substrate concentration required to produce half
maximal velocity of the enzyme catalyzed reaction is termed
Michaelis constant “Km ”.
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Michaelis-Menten kinetic theory of enzyme action
Michaelis-Menten formulated an equation which relates the rate of
enzyme (velocity) catalyzed reaction to substrate concentration:
Vmax /2 [ S ]
Vi =
Km + [S]

Where;
Vi –is the rate (or velocity) of the reaction.
Vmax –is the rate when the enzyme is fully saturated with substrate.
Km - Michaelis constant; is the substrate concentration at
which the reaction rate is half maximal velocity.
 40 Lineweaver-Burk Plot
Represents a linear form of Michaelis-Menten equation and it requires
few points to define Km ( it is the method often used to determine Km
which is expressed as molarity or moles/L).
41 Importance of Km
1- Km is a constant for a particular enzyme under standardized
conditions.

2- Km values are used practically in enzyme assay.

3- Km and Vmax can be affected by pH, temperature and other factors.

4- Km denotes enzyme affinity for its substrate. The higher the

Km the lower the enzyme affinity for its substrate.

5- Km permits evaluation of the inhibitor type (explained later).

6- Isoenzymes differ in their Km .

 42 Enzyme Inhibition
 Enzyme inhibition is one way of regulating enzyme activity. Most
therapeutic drugs function by inhibition of a specific enzyme.

 In the body some of the processes controlled by enzyme inhibition

are blood coagulation, blood clot dissolution (fibrinolysis) and
inflammatory reactions.

Types of inhibitors:
1. Competitive inhibition.
2. Non-competitive inhibition.
3. Uncompetitive inhibition.
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Competitive inhibition:
 Occurs at the substrate binding site.

 The inhibitor is a structural analogue of the substrate, so both are

competing for binding at the enzyme active site.

 Succinate and malonate are 2 structural analogues. So malonate

blocks the action of succinate dehydrogenase on succinate.

 Allopurinol is a competitive inhibitor for xanthine oxidase and used

to treat Gout.
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Example: Lead---forms covalent
bond with sulfhydryl gp of
cysteine in protein
 C. Uncompetitive inhibitors: 50
In this case the inhibitor have no affinity for free enzyme.

The inhibitor (I) bind to [ES] complex ESI

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 52 Enzyme regulation
1-regulation of enzyme quantity:
The amount of enzyme may be
 increased by increasing the rate of synthesis.
 Decreased by decreasing the rate of degradation.

A- Regulation by induction
 Induction of synthesis of a particular enzyme.
 The effector is called inducer (substrate, Hormone).
B-Regulation by repression:
 Number of enzyme molecules decreased by repression.
 The effector is called repressor.
 2-Regulation of catalytic activity 53
Regulatory enzymes:
Control of metabolic pathway may be accomplished by modulation of
key enzymes---catalyse the first step of metabolic sequence (rate-
limiting step) that control the overall pathway.

A-Allosteric regulation:
 Regulate key enzyme.
 Allosteric enzymes oligomeric proteins (more than one subunit).
 Allosteric enzymes posses 2 sites:
i-Catalytic site (active site).
ii- allosteric site-where allosteric modifier bind.
 Binding causes conformational changes in the enzyme which can
either increase (positive allosteric modifier) or decrease (negative
allosteric modifier).
Feedback inhibition: 54
The end product of metabolic pathway results in allosteric
inhibition of the first enzyme in the pathway.
B-Covalent Modification 55
 Covalent modification ----Addition of group to the enzyme by covalent
bond or removal of group by cleaving the covalent bond.

 Covalent modification include phosphorylation and dephosphorylation,

acetylation and deacetylation, methylation and demethylation.

 In mammals phosphorylation and dephosphorylation.

 Phosphoylation (OH group of -----Kinases(ATP).

 Dephosphorylation-----Phosphoprotein phosphatase.

 Glyogen synthase +P inactive (Glycogen synthesis)

 Glycogen phosphorylase+P active (Glycogen breakdown)
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C-Proenzymes: 57
o Is another form of covalent modification but is irreversible.
o Some enzymes are synthesized and secreted in the form of
inactive precursor called zymogen.

o Zymogen Proteolytic cleavage active E + small polypeptide.

o Proteolytic cleavage conformational change reveals

active site.
1- Many proteolytic enzymes of the stomach and pancreas are
secreted as zymogens activated in alimentary canal (this
prevents autolysis of cellular structural proteins). Examples:
Pepsinogen, procarboxypeptidase and trypsinogen.
2- Enzymes of blood clot formation and dissolution secreted
as zymogens and activated when required.
 58 Hormonal regulation of enzymes:

 Regulation via cAMP (cyclic adenosine monophosphate)

which is the second messenger of may hormones (hormone
is the first messenger).

 cAMP activate protein kinases phosphorylate

Target Enzymes
become active or
inactive
(covalent modification)
 59 Enzymes in clinical medicine

The principles of enzymology outlined previously are applied

clinically in 3 ways:

1) Diagnosis and prognosis of diseases.

2) Some enzymes are used as therapeutic agents

3) Enzymes as diagnostic reagents.

 60 Diagnosis and prognosis of diseases

Changes in concentration and activity of plasma enzymes reflect

changes that have occurred in a particular tissue or organ.

Plasma enzymes are of two types:

1-Functional enzymes: synthesized in the liver and present in the blood in
high concentration (perform physiological functions in the blood-----enzymes
associated with blood coagulation..

2-Non-functional plasma enzymes:

 intracellular enzymes present in very low levels in the blood (in
healthy state) and has no function.
 They are released in the plasma as a result of cellular damage (e.g
myocardial infarction &hepatitis).
 61 Enzymes of Diagnostic importance

Enzyme Diagnostic Use

1. Amylase and Lipase Pancreatitis

2. Acid phosphosphatase Prostate cancer

3. Creatine kinase Myocardial infarction and Muscle

diseases
4. Aspartate transaminase Myocardial infarction and
(AST) hepatitis

5. Alanine aminotransferase Viral hepatitis

6. Alkaline phosphatase Bone & Liver diseases

 60 Isoenzymes in diagnosis

 Isoenzymes are different molecular forms of the

same enzyme (differ in amino acid sequence).
 Synthesized by different tissues.
 Isoenzymes catalyze the same reaction.
 They migrate differently in electrophoresis
(because they contain different numbers of charged
amino acids).
 They are made of different subunits.
 Isoenzymes of clinical application include: Lactate
dehydrogenase (LDH) and Creatine Kinase (CK).
Lactate dehydrogenase (LDH): 61
 Tetrameric enzyme formed by combination of 2 subunits: H
(Heart) and M (Muscle):
Type Subunit Tissue of
origin
LDH-1 H4 Heart muscle

LDH-2 H3M1 RBCs

LDH-3 H2M2 Brain

LDH-4 H1M3 Liver

LDH-5 M4 Muscles

 Total LDH is increased in hepatocellular damage, leukemia and

hemolytic anemia In Myocardial infarction total LDH as well as
LDH-1 increased.
 62 Creatine Kinase (CK)
 CK is a dimer having 2 subunits : B for brain and M for muscles:
CK-1---BB-brain, CK-2---MB-heart and CK-3---MM-muscles.

 In myocardial infarction (MI): both CK and LDH increased

 CK increased within 4-6hrs after chest pain and return to normal

within 3days.

 LDH return to normal within prolonged time.

 CK is used for early detection of MI and LDH for follow up of MI.

 63 Enzymes as therapeutic agents

1. Streptokinase: prepared from streptococcus and used in

clearing blood clots in MI. Sterptokinase activates
plasminogen forming plasmin cleaves fibrin into
several soluble components.

2. Asparaginase: used in adult leukemia. Decreases

asparagine level which is needed for tumor cells.
 Enzymes as diagnostic reagents.

Determination of
 Glucose oxidase glucose estimation
 Uricase uric acid
 Urease Urea
 Cholesterol oxidase cholesterol
 Lipase triglycerides

Enzymes used in ELISA ---technique used.

-THE END-