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Enzymes
• Catalyst: A substance that ↑ or ↓ the rate of a chemical reactions without consumed
or affect the end point of the reaction.
• Enzymes:
- Thermo-labile
- Organic catalyst
- Needed in traces in the body
- remain unchanged during the reaction
Characters:
1- Protein in nature, forming colloids of high molecular weight
2- highly specific
3- Produced by living cells but can act outside them
4- acts intra- or extra-cellular

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Enzymes
• Enzymes Terminology: enzymes are named
according to the following systems
1- For hydrolases: name of the substrate (substance
acted upon the enzyme) + ase e.g. urease,lipase,
sucrase, lactase.
2- For other enzymes: name of substrate +
mechanism of action (succinate dehydrogenase)
that removes hydrogen from succinic acid.

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Chemical nature of enzymes
• Enzymes are protein in nature, they are divided
chemically into:
1- Simple protein: consisting wholly of proteins (pepsin,
amylase, lipase)
2- Conjugated protein: (Holoenzymes)
a. Protein part (apoenzymes) thermo-labile, Non-
dialiasable
b. Non-ptn part: Loosely attached (coenzyme (or firmly
attached (prothetic group(

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Chemical nature of enzymes

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Prothetic & coenzyme
Coenzyme Prothetic group
Organic Inorganic
Loosely attached to the protein Firmly attached
and comes in contact during the
activity of the enzyme
Thermo-stable Thermo-liabile
Dialyzable Non-dialyzable
Usually contain vitamin B Usually metal Cu, Fe, Zn
as a part of their structure
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Examples of coenzymes
• NAD: nicotinamide adenine dineuclotide. Nicotinamide + adenine + 2
ribose + 2 phosphoric acid. It acts as a hydrogen carrier.
• NADP: nicotinamide adenine dineuclotide phosphate. Nicotinamide +
adenine + 2 ribose + 3 phosphoric acid. It acts as a hydrogen carrier.
• CoQ: (ubiquinone): it is the quinone used in electron transport and oxidative
phosphorylation.
• CoASH: it is the coenzyme responsible for activation of fatty acids.
• FMN: flavin mononeuclotide. Flavin + ribitol +phosphoric acid. It acts as a
hydrogen carrier.
• FAD: favin adenine dineuclotide. Riboflavin + phosphate + AMP. It acts as
a hydrogen carrier.

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Mechanism of enzyme action
• Lowering of activation energy
- for a reaction to occur, the substrate should be placed
at a high energy level i.e. activation energy which is to
be supplied to the reaction.
- Enzymes are lower this activation energy.
- Example: activation energy required for hyrolysis of
sucrose is 26,000 calories/mol, when sucrase enzyme
is used the activation energy is reduced to 9,000
calories/mol.

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Mechanism of enzyme action
• Michaelis-Menton theory (enzyme-substrate
complex)

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Mechanism of enzyme action
• Active center:
Certain areas of the enzyme will be functionally
more important and named the active site. This
area can unite with the substrate forming an E-S-
complex.

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Mechanism of enzyme action
• Lock and key theory (Fisher’s Template theory)

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Mechanism of enzyme action
• Some enzymes are secreted in an inactive form
(extracellular enzymes), these are known as zymogen
or proenzymes. The active site of these zymogen are
masked by polypeptide chains. These proenzymes
must be activated first by removal of these polypeptide
chains by either:
HCl
pH change : pepsinogen → pepsin
trypsin
Autocatalysis: trypsinogen → trypsin
Kinases: trypsinogen → trypsin
kinases

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Factors affecting enzyme activity
• Nature of the enzyme (isoenzyme or isozyme)
- Catalyze the same reaction.
- Differ in structure, properties, and the rate of
enzymatic activity.
- The best example is lactate dehydrogenase
(LDH), which is by electrophoresis is separated
into 5 isoenzymes.

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Lactate dehydrogenase (LDH)
• Consists of 4 polypeptide chains )tetramers) of 2 types
H, M
• Type I: HHHH→ heart
Type II: HHHM
Type III: HHMM
Type IV : HMMM
Type IV : MMMM → muscle and liver

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Factors affecting enzyme activity
• Concentration of the substrate: if the concentration of
substrate is increased and all other conditions are kept
constant, the rate of enzyme action increase up to a certain
point.

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Factors affecting enzyme activity
• Concentration of the enzyme: The velocity of reaction
directly proportion to concentration of enzyme up to a
certain limit.

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Factors affecting enzyme activity
• Temperature: The optimum temperature for an enzyme is that
temperature at which the greatest amount of substrate is changed in
unit time. Above this temperature → ↓ enzyme Activity. At about
70ºC the enzyme is destroyed, due to denaturation (ptn. In nature

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Factors affecting enzyme activity
• pH: Affect ionization of a.a. in the active site of the
enzyme &substrate.
• Each enzyme has an optimum pH at which its activity is the best
- Salivary amylase 6.8 Pancreatic lipase 8.0

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Factors affecting enzyme activity
• Temperature: Every enzyme has an optimum
temperature above this temperature → denaturation (↓
enzyme Activity)
• Human (37-40˚) plants (65-70 ˚)

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Factors affecting enzyme activity
Effect of hormones:
- Insulin +→ glucokinase ← - glucocorticoids
- Steroid hormones → ↑ rate of synthesis of many enzyme
Enzyme activators:
some enzymes are secreted inactive → zymogen or
proenzymes (proteolytic enzymes)
- pH: pepsinogen → by HCl pepsin
- Autoactivation

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Factors affecting enzyme activity
• Enzyme inhibitors:
A. Non specific inhibitors; affect a wide variety of enzymes
1) Agents which precipitate or denaturate ptn.
2) Sulphahydryl inhibtors:
- Oxidizing agents as K ferricynaide → oxidizing SH → S
—S
- Alkalyating agents as iodoacetic acid → block SH
- Salts of heavy metals (Hg, Pb)

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Factors affecting enzyme activity
• Enzyme inhibitors:
B. Specific inhibitors: acts only on one enzyme
1. Competitive, reversible, or substrate inhibitors:
- Both substrate and inhibitor are structurally similar.
- Both compete for the active site of the enzyme.
- The inhibitor combine reversibly with the active site
- The inhibitor can be reversed by increasing substrate
concentration

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Competitive inhibitors
• malonate competes with succinate for active
site on succinate dehydrogenase in TCA cycle

Fumarate
malonate Succinate

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Competitive inhibitors
• e.g. sulphonamides (e.g. prontosil) act as
antibiotics by inhibiting folic acid synthesis
(folic acid is important in bacterial cell walls)

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Competitive inhibitor
• Dicumarol competes with vitamin K for
epoxide reductase → ↓ vitamin K synthesis→
used as anti-coagulant

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Competitive inhibitor
• Allopurinol competes with hypoxanthine for
xanthine oxidase enzyme→ ↓ uric acid →
used in treatment of gout

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Non-competitive inhibitors
• No structural similarity between the substrate
and inhibitors.
• No competition
1- Allosteric modifiers
Reversible, when the modifier is consumed (not
by increasing the substrate concentration) →
return to the normal

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Allosteric modification
• Are small organic molecules which have regulatory role
on enzyme activity.
• The inhibitor binds with the apoenzyme (allosteric site)
far from the active site

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Allosteric modification
• Mechanism: The reversible binding of the allosteric
inhibitor produce conformational change in the apo-
enzyme, so binding of the substrate to the active site
become difficult.
• Example; ATP & citrate are allosteric inhibitors of PFK1.
- G-6-P is allosteric inhibitor of
hexokinase.

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Allosteric modifiers
• Sometimes, binding of the allosteric modifiers
to the apo-enzyme produce conformational
change, making the active site more fit for the
substrate (Allosteric activators).
• Example; AMP, ADP are allosteric activators
of PFK1

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Non-competitive inhibitors
2- Feedback inhibitors
Product of reactions inhibits activity of an enzyme in the
biosynthetic pathway (by binding with the enzyme or
repress the gene coding the enzyme)

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Non-competitive inhibitors
3- inhibitors blocking the coenzyme or prothetic group:
Agents which block the coenzyme will stop enzyme action
i.e. phenylhydrazine block aldhyde group of PLP
(coenzyme for transaminase)
Agents which block the prothetic group will stop enzyme
action i.e. cynaide and CO block iron of heme in
cytochrome oxidase.
Fluoride block enzyme requiring Mg

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Non-competitive inhibitors
4- Antienzymes & antibodies:
Antienzymes: as ascaris worm secrete anti-pepsin and
anti-trypsin to prevent digestion of worm.
Antibodies: if enzymes are injected, the immune system
of the body produce antibodies to inactivate these
enzymes.
Concentration of co-factors:
Rate of chemical reaction is directly proportional to the conc. Of
co-factors e.g. coenzyme or prothetic group.

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Coenzyme & H2 carrier
• Coenzymes
Nicotinamide nucleotides: NAD, NADP
Flavin nucleotides: FMN, FAD
• H2 carrier
Nicotinamide nucleotides
Flavin nucleotides
Glutathione: glutamic acid + cystiene + glycine

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Importance of enzymatic
determination in clinical diagnosis
• Functional plasma enzymes:
- has a function in plasma
- Substrates are present at ↑ concentration
- e.g. lipoprotein lipase & blood clotting enzyme.
 Non-Functional plasma enzymes:
- no specific function
- no substrate to act
- Derived from normal breakdown and re-synthesis
- Present in low concentration
- If the cells are destroyed by a disease↑ (very helpful in
diagnosis)

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Enzymes of medical importance
• Serum amylase: ↑in parotitis, acute pancrititis, pancreatic cancer.
• Serum lipase: ↑ in acute pancrititis & pancreatic cancer.
• Serum trypsin: ↑ in pancreatic disease.
• Serum alkaline phosphatase: ↑ in obstructive jaundice, bone disease,
rickets, osteogenic cancer, hyperparathyroidism, and malignancy.
• Serum acid phosphatase: ↑ in prostatic cancer.
• Serum LDH: ↑ in acute MI, liver and muscle disease.
• Serum CK: ↑ in brain, heart, and muscle disease.
• Serum transaminases: (AST in liver disease & MI), ALT (liver
disease)

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Regulation of enzyme activity
• Change the absolute amount of the enzyme
• Change the catalytic activity of the enzyme
• Compartmization of the enzyme
• Allosteric regulation
• Covalent modification

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Regulation of enzyme activity
• Change the absolute amount of the enzyme
a. By controlling enzyme synthesis: induction or repression
Inducer: substances which stimulate gene expression as
substrate or hormones.
Repressor: substances which inhibit gene expression as product
or hormones.
b. By controlling enzyme degradation.
• Change the catalytic activity of the enzyme
By temperature, pH, co-factors

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Regulation of enzyme activity
• Compartmization of the enzyme
The different cells or organelles are separated by membranes, the
transport across these membrane are under control and
regulation. Enzymes of urea cycle, part in the cytosol and part
in the mitochondria (to facilitate regulation of these pathway).
• Allosteric regulation
By allosteric activators or inhibitors.
• Covalent modification
By phosphorylation and dephosphorylation.

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Classification of enzyme
• According to site of action
Intracellular: produced by cells and act within cells.
Extracellular: produced by cells and act outside cells. May be secreted active
(salivary & pancreatic lipase) or inactive (pepsinogen & trypsinogen by
pH and autoactivation).

• According to enzyme specificity:


1. Absolute specificity: act on ONE substrate (sucrose & sucrase)
2. Relative specificity: act on a GROUP of closely related substrates which
are similar in structure and bonds (lipase hydrolyze ester-linkage of TAG
containing different FA, amylase hydrolyze glucosidic linkage in starch,
dextrin &glycogen)

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Classification of enzyme
• According to enzyme specificity:
3. Stero- or optical specificity: act on ONE of two isomers (L-
a.a. oxidase acts on L-amino acid) except racemase which
catalyzes interconversion between L- and D- forms.
4. Dual specificity: The enzyme act on TWO different
substrates (xanthine oxidase acts on hypoxanthine and
xanthine).
5. Group or structural specificity: Specificity to BONDS, its
POSITION & CHEMICAL GROUPS surrounding them
(carboxypeptidase acts on free COOH of the polypeptide
chain).

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According to the function

Subgroup Main group


Dehydrogenase Oxido-reductase
Oxidases (H2O, H2O2)
Hydroperoxidase i.e. catalase or
peroxidase
Oxygenase
Transphosphorylases (kinase) Transferases
Transaminase
Transmethylase
Peptidases (Endo- or Exo) Hydrolyses
Estrase
Amidases
Phosphorylases
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According to the function
Subgroup Main group

Dehydratases
Lyses
Decarboxylases
Desulfhydrases
Carboxylases
Ligases
Acyl CoA synthathase

Isomerases

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