Enzymes are protein catalysts that accelerate chemical reactions in living organisms. They facilitate reactions by lowering the activation energy needed. Enzymes achieve specificity through their active sites, which are complementary in shape and chemical properties to their substrates. Factors like temperature, pH, and inhibitors can impact an enzyme's activity. There are several mechanisms of enzyme action and regulation, including competitive and non-competitive inhibition, as well as allosteric regulation through effectors binding at distinct sites. Precise control of enzymes is crucial for metabolic processes in cells and organisms.

1. Kapenda Joel

2. INTRODUCTION TO ENZYMES
 Enzymes are biologic polymers that catalyze the chemical reactions which
make life as we know possible.
 The presence and maintenance of a complete and balanced set of enzymes is
essential for ;
- the breakdown of nutrients to supply energy and chemical building blocks;
- the assembly of those building blocks into proteins, DNA, membranes, cells,
and tissues; and
- the harnessing of energy to power cell motility and muscle contraction.
 With the exception of a few catalytic RNA molecules, or ribozymes, the vast
majority of enzymes are proteins.
 Their catalytic activity depends on the integrity of their native protein
conformation.
 Deficiencies in the quantity or catalytic activity of key enzymes can result from
genetic defects, nutritional deficits, or toxins.

3.  Substances on which enzymes act to convert them into products are called
substrates.
 Enzymes have immense catalytic powers and accelerate reactions at least a
million times by reducing the energy of activation.
 For chemical reaction to take place, the reacting molecules are required to gain
a minimum amount of energy, called energy of activation.
 Few enzymes are simple proteins while some are conjugated proteins.
 In such enzymes the non-protein part is called prosthetic group or coenzyme
and the protein part is called apoenzyme.
 When many different enzyme catalyzing sites are located at different sites of
the same macromolecule, it is called multienzyme complex. Examples: fatty
acid synthetase, carbamoyl phospahte synthetase II, pyruvate dehydrogenase,
etc.
 The complex becomes inactive when it is fractionated into smaller units each
bearing individual enzyme activity.

4. COENZYMES
 Certain enzymes require non-protein organic coenzymes for the activity.
 Prosthetic groups are distinguished by their tight, stable incorporation into a
protein’s structure by covalent or non-covalent forces.
 Examples include Biotin, tertrahydrofolate, NAD+, NADP, Co, Cu, Zn etc.
 Enzymes that contain tightly bound metal ions are termed metalloenzymes.
COFACTORS
 Cofactors serve functions similar to those of prosthetic groups but bind in a
transient, dissociable manner either to the enzyme or to a substrate such as
ATP.
 Cofactors must be present in the medium surrounding the enzyme for catalysis
to occur.
 Enzymes that require a metal ion cofactor are termed metal-activated
enzymes.

5. NOMENCLATURE AND CLASSIFICATION OF ENZYMES
 In order to have uniformity and unambiguity in identification of enzymes, the
Internation Union of Biochemistry (IUB) adopted a nomenclature sysytem
based on chemical reaction type and reaction mechanism.
 According to this system, enzymes are grouped into six main classes. They are:
1. Oxidoreductase: catalyze oxidations and reductions of their substrates, e.g.
alcohol dehydrogenase, lactate dehydrogenase.
2. Transferase: catalyze transfer of particular group from one substrate to
another, e.g. hexokinase, aspartate and alanine transaminase (AST/ALT).
3. Hydrolase: bring about hydrolysis, e.g. glucose-6-phospatase, pepsin, trypsin.
4. Lyases: addition of groups to double bonds, or formation of double bonds by
removal of groups, e.g. fumarase, arginosuccinase.
5. Isomerases: transfer of groups within molecules to yield isomeric forms, e.g.
UDP-glucose, epimerase.
6. Ligases: catalyze joining together two substrates coupled with ATP hydrolysis,
e.g. DNA ligase, glutamine synthetase.

6. SPECIFICITY OF ENZYMES
 An important property of enzyme is their specificity. Specificity is of 4 different
types;
1. Optical specificity:
 There can be many optical isomers of a substrate, but only one of the isomers
acts as a substrate for the enzyme activity.
2. Reaction specificity:
 An enzyme can catalyze only a single type of reaction.
 A substrate can undergo many reaction, each reaction catalysed by different
enzymes.
3. Substrate specificity:
 This means that certain enzymes are specific for a certain substrate.
 Substrate specificity is of two type; group dependent and bond dependent.

7.  Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
- E.g. Trypsin hydrolyses the residues of only lysine and arginine, chymotrypsin
hydrolyses residues of only aromatic amino acids.
 Bond specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.
- E.g. Proteolytic enzymes, glycosidases and lipases act on peptide, glycosidic
and ester bonds respectively.

8. MECHANISM OF ENZYME ACTION
 According to most acceptable hypothesis, enzyme molecule (E) first
combines with substrate molecule (S) to form an enzyme-substrate (ES)
complex which further dissociates to form product (P) and enzyme (E).
 Enzyme once dissociated from ES complex is free to combine with another
substrate and form product.
 The ES complex is an intermediate or transient complex held together by weak
non-covalent bonds such as H-bonds, Van der Waals forces, hydrophobic
interactions.
 The site at which the substrate can bind to the enzyme with extreme specificity
is called active site or catalytic site.
 The active site is made up of several amino acids that come together as a result
of folding of secondary and tertiary structures of the enzyme.

9. MODELS OF ENZYME-SUBSTRATE COMPLEX FORMATION
1) Template or Lock and Key Model
 This model states that the active site already exists in proper conformation
even in the absence of the substrate.
 The active site provides a rigid, pre-shaped template fitting with the size and
shape of the substrate molecule.
 Substrate fits into the active site as key fits into lock, hence called lock and key
model.
 Model cannot explain change in enzyme activity in presence of allosteric
modulators.
2) Induced Fit or Koshland Model
 Important feature of this model is the flexibility of active site region.
 According to this, the substrate during its binding induces conformational
changes in the active site to attain the final catalytic shape and form.

10.  This model explains;
- enzymes become inactive on denaturation
- saturation kinetic
- competitive inhibition
- allosteric modulation

11. ENZYMES EMPLOY MULTIPLE MECHANISMS TO FACILITATE
CATALYSIS
Four general mechanisms:
Catalysis by Proximity
 For molecules to react, they must come within bond forming distance of one
another.
 The higher their concentration, the more frequently they will encounter one
another and the greater will be the rate of their reaction.
 When an enzyme binds substrate molecules in its active site, it orients the
substrate molecules spatially in a position ideal for them to interact.
Acid-Base Catalysis
 The ionizable functional groups of aminoacyl side chains and of prosthetic
groups contribute to catalysis by acting as acids or bases.
 Acid-base catalysis can be either specific or general.

12.  In specific acid or specific base catalysis, the rate of reaction is sensitive to
changes in the concentration of protons but independent of the concentrations
of other acids or bases present in solution or at the active site.
 Reactions whose rates are responsive to all the acids or bases present are said to
be subject to general acid or general base catalysis.
Catalysis by Strain
 Enzymes that catalyze lytic reactions typically bind their substrates in a
conformation slightly unfavorable for the bond that will undergo cleavage.
 The resulting strain stretches or distorts the targeted bond, weakening it and
making it more vulnerable to cleavage.
Covalent Catalysis
 This catalysis involves the formation of a covalent bond between the enzyme
and one or more substrates.
 The modified enzyme then becomes a reactant.
 The chemical modification of the enzyme is, however, transient.
 Covalent catalysis is particularly common among enzymes that catalyze group
transfer reactions.

13. The Active Sites of Enzymes Have Some Common Features
1. The active site is a three-dimensional cleft formed by groups that come from
different parts of the amino acid sequence.
2. The active site takes up a relatively small part of the total volume of an
enzyme.
3. Active sites are clefts or crevices.
4. Substrates are bound to enzymes by multiple weak attractions.
5. The specificity of binding depends on the precisely defined arrangement of
atoms in an active site.

14. FACTORS AFFECTING ENZYMES
Temperature
pH
Enzyme concentration
Substrate concentartion
Inhibitors

15. Temperature
 As the temperature rises, reacting molecules have more and more kinetic
energy. This increases the chances of a successful collision and so the rate
increases.
 Each enzyme is most active at a specific temperature, called optimum
temperature.
 This optimal temperature is usually around human body temperature (37.5 o C)
for the enzymes in human cells.
 Above this temperature the enzyme denatures since it is at higher
temperatures intra- and intermolecular bonds are broken as the enzyme
molecules gain even more kinetic energy.
 The Q10 or temperature coefficient is a measure of
the rate of change of a biological or chemical system as
a consequence of increasing the temperature by 10 °C.

16. pH
 pH at which its activity is greatest is called the optimal pH.
 Extreme pH levels will cause denaturation.
 The active site is distorted and the substrate molecules will no longer fit.
 Small changes in pH above or below the optimum do not cause a permanent
change to the enzyme, since the bonds can be reformed.
 H+ and OH- Ions are charged and therefore interfere with Hydrogen and
Ionic bonds that hold together an enzyme, since they will be attracted
or repelled by the charges created by the bonds.
 This interference causes a change in shape of the enzyme, and importantly,
its active site.

17. Enzyme Concentration
 Rate of enzyme activity is directly proportional to
enzyme concentration as long as the substrate
concentration is in excess.
Substrate Concentration
 Increasing substrate concentration increases the rate of reaction. This is
because more substrate molecules will be colliding with enzyme molecules,
so more product will be formed.
 After a certain concentration, any increase will have no
effect on the rate of reaction, because enzymes will
effectively become saturated.
 The enzyme-substrate complex has to dissociate before
the active sites are free to accommodate more substrate.

18. Inhibitors
 Enzyme inhibitors are substances which alter the catalytic action of the enzyme
and consequently slow down, or in some cases, stop catalysis.
 Whenever the active site is not available for binding of the substrate the
enzyme activity may be reduced.

19. ENZYME INHIBITION
 The chemical substances which inactivate enzymes are called inhibitors and
the process is called enzyme inhibition.
 Enzymes catalyze virtually all cellular processes, enzyme inhibitors are among
the most important pharmaceutical agents known.
 For example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the
first step in the synthesis of prostaglandins, compounds involved in many
processes, including some that produce pain.
 Three major groups of inhibition:
1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition

20. Reversible inhibition.
 When the active site or catalytic site is occupied by a substance other than the
substrate, its activity is inhibited.
 One common type of reversible inhibition is called competitive inhibition.
 A competitive inhibitor [I] competes with the substrate for the active site of an
enzyme.
 While the inhibitor occupies the active site it prevents binding of the substrate
to the enzyme.
 Many competitive inhibitors are compounds that resemble the substrate and
combine with the enzyme to form an EI complex, but without leading to
catalysis.
 Combinations of this type will reduce the efficiency of the enzyme.
 Because the inhibitor binds reversibly to the enzyme, the inhibition can be
overcome by adding more substrate.
Clinical Significance:
 Medical therapy based on competitive inhibition is used to treat patients who
have ingested methanol, a solvent found in gas-line antifreeze.

21.  The liver enzyme alcohol dehydrogenase converts methanol to formaldehyde,
which is damaging to many tissues especially eyes.
 Ethanol competes effectively with methanol as an alternative substrate for
alcohol dehydrogenase converting it to acetaldehyde.
 This slows the formation of formaldehyde, lessening the danger while the
kidneys filter out the methanol to be excreted harmlessly in the urine.
 Allopurinol is a drug used to treat gout. Uric acid is formed in the body by
oxidation of hypoxanthine by the enzyme xanthine oxidase. Allopurinol acts as
a competitive inhibitor of xanthine oxidase reducing uric acid formation.
 Methotrexate is used in cancer therapy. It’s a structural analog of folic acid. It
inhibits folate reductase and prevents formation of FH4, which in turn inhibits
DNA synthesis.
 Succinylcholine is used as a muscle relaxant. It is structurally similar to
acetylcholine. It competitively binds to post-synaptic receptors.
Acetylcholinesterase cannot hydrolyse them which causes continued
depolarization resulting in muscle relaxation.
 Dicoumarol is used as an anticoagulant, structurally similar to vitamin K and
competitively inhibits vitamin K epoxide reductase, an enzyme that
recycles vitamin K.

22.  Two other types of reversible inhibition, uncompetitive and mixed.
 An non-competitive inhibitor binds at a site distinct from the substrate
active site and binds only to the ES complex.
 If the inhibitor can be removed from its binding site without affecting enzyme
activity, it is called reversible non-competitive inhibition.
 A mixed inhibitor also binds at a site distinct from the substrate active site,
but it binds to either E or ES.
 Non-competitive and mixed inhibition are observed only for enzymes with two
or more substrates.
 These inhibitors bring about changes in the 3D structure of the enzyme
inactivating.

23. Irreversible inhibition
 The irreversible inhibitors are those that bind covalently with or destroy a
functional group on an enzyme that is essential for the enzyme’s activity.
 If non-competitive inhibitor can be removed only at the loss of enzyme activity,
it is known as irreversible non-competitive inhibition.
 Examples of non-competitive inhibitor ;
- iodoacetate inhibiting enzymes like glyceraldehyde 3-phospahte and papin
- heavy metals like silver, mercury
- fluoride inhibits emolase
Clinical Significance
 British anti Lewesite (BAL) is used as antidote for heavy metal poisoning.
Heavy metals inhibit enzymes by reacting with –SH groups. BAL provides –SH
for the heavy metals to act on.
 Disulfiram used in treatment of alcoholism. It inhibits aldehyde
dehydrogenase preventing oxidation of acetaldehyde which accumulates
producing sickening effect leading to aversion to alcohol.

24.  Suicide inhibition is a special type of irreversible non-competitive inhibition
in which the substrate analog is converted to a more effective inhibitor with
the help of the enzyme to be inhibited.
 The new inhibitor formed binds irreversibly with the enzyme.
 Examples include allopurinol, aspirin, 5-fluorouracil.
Allosteric inhibition
 It is a kind of inhibition when the inhibitor binds to the enzyme at a site other
than the active site, sometimes on a different region in the enzyme called
allosteric site.

25. REGULATORY ENZYMES
 Regulatory enzymes exhibit increased or decreased catalytic activity in
response to certain signals.
 These enzymes allow the cell to meet changing needs for energy and for
biomolecules required in growth and repair.
 Allosteric enzymes function through reversible, non-covalent binding of
regulatory compounds called allosteric modulators or allosteric effectors,
which are generally small metabolites or cofactors.
 Some enzymes are stimulated or inhibited when they are bound by separate
regulatory proteins.
 Changes in enzyme-substrate interaction due to the allosteric effects of
regulatory molecules other than the substrate are called heterotropic alloteric
modulation.
 When the binding of substrate enhances the interaction between the allosteric
enzyme and more substrate molecules is called homotropic allosteric effects.

26.  Other enzymes are regulated by reversible covalent modification.
 These include the phosphorylation, adenylation, acetylation, uridylation, ADP-
ribosylation, and methylation of enzymes.
 The covalently attached groups are removed from the enzyme by separate
enzymes.
 Phosphorylation is the most common type of regulatory modification found in
eukaryotes. It is the addition of phosphate group.
 An important example of regulation by phosphorylation is observed in the
enzyme glycogen phosphorylase of muscle and liver.
 Methylation is the addtion of a methyl group to a protein. Enzyme regulation
by methylation can be observed in the methyl-accepting chemotaxis protein of
bacteria.

27.  Feedback regulation is when the product inhibits or activates the enzyme
activity in response to stimuli.
 If the end product becomes available in the environment, it is unnecessary and
wasteful for the cells to continue to produce the product. Cells have the ability
to shut down a pathway when it is not needed.
 In biochemical pathways, the product of one reaction becomes the substrate
for the next reaction.
 When the regulatory enzyme reaction is slowed, all subsequent enzymes
operate at reduced rates as their substrates are depleted.
 After the product has been utilized and its concentration decreased, the
inhibition is relaxed, and the formation of the product resumes.
 Example: Dietary cholesterol restricts the synthesis of cholesterol from acetate
in mammalian tissues.

28. CLINICAL USES OF ENZYMES
 Enzymes are used clinically in three principal ways:
1. 1. As indicators of enzyme activity or enzyme concentration in body fluids
(e.g., serum and urine) in the diagnosis and prognosis of various diseases;
2. 2. As analytical reagents in the measurement of activity of other enzymes or
non-enzyme substances (e.g., substrates, proteins, and drugs) in body fluids;
and
3. 3. As therapeutic agents.

29.  Analysis of the presence and distribution of enzymes and isozymes often aids
diagnosis.
 Isozymes (also known as isoenzymes) are enzymes that differ in amino acid
sequence but catalyze the same chemical reaction.
 An example of an isozyme is glucokinase, a variant of hexokinase.
 Serum levels of a particular enzyme maybe increased by diseases that cause;
a) increase in its rate of release due to necrosis of cell, increase in cell
permeability, increased cell division.
b) decrease in the rate of disposition or excretion due to obstructive jaundic,
renal failure.
 Decreased serum levels can be caused by;
a) decreased production of the enzyme due to genetic defects or acquired
(caused by diseases or malnutition)
b) enzyme inhibition caused by insecticide poisioning.
c) lack of cofactors in pregnancy and cirrhosis.

30. Unit of Serum Enzyme Activity
 Serum enzyme activity is expressed in International Units (IU).
Value of Serum Enzyme Assay
 Assay of the activity of selected serum enzyme or enzymes can provide
information on the nature and extent of a disease.
 Helps in differential diagnosis, i.e. distinguishing of a disease or condition from
others presenting with similar signs and symptoms. Example, helping to
differentiate between myocardial infarction (MI) and pulmonary embolism
both presenting with chest pain.
 Serial enzyme assays are required to ascertain prognosis, i.e. to check progress
of a disease, check response to therapy.
 Helps in early detection of disease, even before symptoms manifest.

31. SERUM ENZYMES IN HEART DISEASES
1. CREATINE PHOSPHOKINASE (CPK)
 Also called creatine kinase (CK).
 Found in high concentration in skeletal muscles, myocardium and brain, not
found in liver , RBCs and kidney.
 In the cells, CK enzymes consist of two subunits, which can be either B (brain
type) or M (muscle type). There are, therefore, three different isoenzymes:
CK-MM, CK-BB and CK-MB.
 CK-MM is expressed in skeletal and cardiac muscle.
 CK-MB is expressed in cardiac muscle, and
 CK-BB is expressed in smooth muscle and in most non-muscle tissues.
 Normal values range from 8 – 150 IU/L

32. 2. SERUM GLUTAMATE OXALOACETATE TRANSAMINASE (S-GOT)
 Also known as aspartate transaminase (AST).
 Very high concentration in myocardium.
 Normal values range from 10 – 40 IU/L.
 Levels > 350 IU/L usually fatal (due to massive MI)
 Levels > 150 IU/L associated with high mortality
3. LACTATE DEHYDROGENASE (LDH)
 Found extensively in body tissues, such as blood cells and heart muscle.
 Functional lactate dehydrogenase are homo or hetero tetramers composed of
M and H protein subunits:
i. LDH-1 (4H)—in the heart and in RBC
ii. LDH-2 (3H1M)—in the reticuloendothelial system
iii. LDH-3 (2H2M)—in the lungs
iv. LDH-4 (1H3M)—in the kidneys, placenta, and pancreas
v. LDH-5 (4M)—in the liver and striated muscle

33.  Normal range is 60 – 250 IU/L
 LDH elevation may persist for more than a week after CPK and AST levels have
returned to normal.
 It is relatively non-specific for MI since its widespread in the body.
 Coexistent diseases in other organs can cause elevation, such as pulmonary
infarction, renal necrosis, muscle diseases, hemolysis of RBCs.
4. CARDIAC TROPONINS
 Troponins are of 3 types:
i. Troponin –C (Clacium binding); non-cardiac
ii. Troponin I; Cardiac T1 (CT 1)
iii. Troponin T; Cardiac T (CTT)
 Normal values less than 1.5 mg/L

35. SERUM ENZYMES IN LIVER DISEASES
 The serum enzymes used in assessment of liver function are divided into two
categories:
1. markers used in hepatocellular necrosis and
2. markers that reflect cholestasis.
 Alanine aminotransferase (ALT) and aspartate aminotransferases (AST) are
markers for hepatocellular necrosis.
 Normal range of ALT is 7 – 55 IU/L
 Normal range of AST is 8 – 48 IU/L
 Serum enzymes used as markers of cholestasis include alkaline phosphatase
(ALP), 5'-nucleotidase, and y-glutamyl transferase.
 Normal range of ALP is 40 – 140 IU/L.
 Normal range of GGT is 9 – 48 IU/L.
 Normal range of 5’-nucleotidase is 1 – 17 IU/L.

36. ENZYMES USED AS LAB REAGENTS
 Some enzymes are used in the estimation of biomolecules in serum.
Examples:
 Glucose oxidase enzyme is used for estimation of glucose in body and body fluid.
 Uricase is used for the estimation of serum uric acid.
 Urease is used in the estimation of urea in blood and body fluids.
THREAPEUTIC USES OF ENZYMES
 Enzymes have been used as drugs to treat various disorders.
Examples:
 Streptokinase used to treat MI. Used to dissolve clots in the arteries of heart wall.
 Collagenase used to treat skin ulcers, causes collagen hydrolysis.
 DNAse used in treatment of Cystic Fibrosis (CF). DNAse hydrolyses extracellular
DNA responsible for CF.
 Uricase used to treat gout. Converts uric acid to allantoin.

37. ISOENZYMES
 Isozymes (also known as isoenzymes) are enzymes that differ in amino acid
sequence but catalyze the same chemical reaction.
 In biochemistry, isozymes are isoforms (closely related variants) of enzymes.
 LDH exists as at least five different isozymes separable by electrophoresis.
 All LDH isozymes contain four polypeptide chains, each type containing a
different ratio of two kinds of polypeptides.
 The M (for muscle) chain and the H (for heart) chain are encoded by two
different genes.
 Different isoenzymes are often organ-specific and their determination may
improve the specificity of enzyme tests.
 LDH1 (HHHH) – Present in Heart and Erythrocyte
 LDH2 (HHHM) – Present in Heart and Erythrocyte
 LDH3 (HHMM) – Present in Brain and Kidney
 LDH4 (HMMM) – Present in Skeletal Muscle and Liver
 LDH5 (MMMM) – Present in Skeletal Muscle and Liver