This document provides an overview of enzymes, including their classification, properties, mechanisms of action, kinetics, and regulation. It discusses how enzymes are classified into six main categories based on the type of reaction they catalyze. Key properties of enzymes include their specificity, ability to catalyze reactions without being consumed, and presence of cofactors. Enzymes lower the activation energy of reactions, allowing them to proceed more rapidly. Their activity can be regulated by various mechanisms and is dependent on factors like substrate concentration, temperature, and pH. The kinetics of enzyme-catalyzed reactions are described by the Michaelis-Menten equation. Inhibitors can interfere with enzyme activity through competitive, noncompetitive, or uncompetitive inhibition.

1. ENZYMES
Dr. Solomon Genet
Department of Biochemistry
School of Medicine
AAU

2. Lecture Overview
(1) Introduction to Enzymes
(2) Classification of enzymes
(2) How Enzymes Work
(3) Catalytic Groups
(4) Properties of enzymes
(5) Enzyme kinetics
(6) Enzymes in drug synthesis
(7) Enzymes in clinical diagnosis

3. Definitions
Enzymology: is the study of enzymes
Catalyst : a substance that increases the
rate of a reaction without itself being
consumed (changing)
- Enzymes: are biological catalysts
- All enzymes are proteins except for few
RNA that have catalytic property
Some enzymes require co-factors (e.g.
inorganic ions)

4. Definition …
• Coenzyme – a complex organic or
metalloorganic molecule (may contain a
vitamin as a component)
• Prosthetic group – coenzyme or metal
ion that is very tightly bound (sometimes
covalently)
• Holoenzyme – enzyme with a cofactor
• Apoenzyme – the protein part of a
holoenzyme

5. Classification of Enzymes
• 6 Classes of Enzymes are recognized by
IUBMB
1) Oxidoreductases: transfer of electrons
2) Transferases: Catalyze transfer of groups
3) Hydrolases: Hydrolysis reactions
4) Lyases: Addition of groups to double bonds or
formation of double bonds by group removal
5) Isomerases: Transfer of groups within a
molecule to yield isomeric forms
6) Ligases: Bond formation 9C-C, C-O,C-S, C-N

6. Enzyme Nomenclature
use E.C. Enzyme Commision Number
Eg: ATP + D-glucose → ADP + D-glucose-6-P
ATP:glucose phosphotransferase
Trivial name: Hexokinase
E.C. 2.7.1.1 (Commision number)
(2) – denotes class name-transferase
(7) – denotes subclass-phospotransferase
(1) – denotes phosphotransferase with a hydroxyl group
as an acceptor
(1) denotes D-Glucose as the phosphoryl group
acceptor

7. Properties of Enzymes
1) Specificity: Enzymes are very specific to their
substrates (Broad or absolute)
Eg. Hexokinase has broad specificity bcs it acts
on hexose sugars (Glucose, Mannose,
Fructose)
Glucokinase is specific for only Glucose
Enzymes are also stereospecific hence may
act on only one stereoisomer.

8. Properties of Enzymes…
2) Turnover number: Enzymes have high
turnover number i.e. an enzyme molecule can
act on many substrate molecules in a reaction.
This simply refers to their catalytic power.
Enzymes do not themselves take part in
biological reactions and hence are
reproducible and can be recycled during
catalysis

9. Properties of Enzymes
3)Regulation: Enzyme activities can be regulated
in many ways
a. Allosteric regulation
b. Feedback inhibition
c. Zymogen activation
d. Covalent modification

10. Properties…
4) Active site: a cleft or pocket formed as the
enzyme folds
• Contains amino acid side chains that form
complementary structure to the substrate
• Site at which substrate binds
• Mostly polar amino acids like Cys, Ser, Thr, Tyr
are found at the active site.
Sometimes amino acids triads are found (Serine
proteases)

11. Properties…
5) Distribution: enzymes are found distributed in
different tissues.
They are found in specific organelles and
compartmentalization helps specific reaction to
be undergone without interference in a
controlled way.
Some enzymes are tissue specifically expressed,
which makes them markers for disease
diagnosis.

12. Properties…
6) Cofactors: Enzymes require cofactors needed
for catalytic activity. These cofactors may be
inorganic metal ions (Mn, Mg, Fe, Se,Zn etc) or
organic groups called coenzymes.
Coenzymes are derived from vitamins [Eg TPP
from thiamine (B1), PLP (B6), NAD (Niacin),
FAD (Riboflavin) etc]
7) Isoenzymes: Some enzymes are distributed
between tissues as isoenzymes (enzymes that
catalyze the same type of reaction and have
structural similarity)

13. MECHANISM OF ACTION
• Enzymatic catalysis of biological reactions is
essential to living systems.
• Enzymes hasten chemical reactions or they
affect rate of a reaction without affecting the
equilibria.
E + S ES → P + E
• Where E = enzyme, S= substrate, ES= enzyme
substrate complex, P= product
• NB: the substrate attaches to enzyme active
site and forms ES complex before
transformation to product.

14. Models of Enzyme-Substrate interaction
• Two models have been suggested for the
interaction of enzymes with their substrates
1) Lock and Key model: It is assumed that the
substrate and the active site have structural
similarity as a key fits its padlock.
2) Induced fit model: Approaching of the
substrate to an enzyme active site brings
about a conformational change on the active
site. This model better explains properties of
allosteric enzymes.

15. MECHANISM OF ACTION…
• Mechanism of enzyme action can be viewed
in two perspectives
1) They provide an alternate energetically
favorable (energy minimal) pathway compared
to uncatalyzed same reaction.
2) The active site chemically facilitates catalysis
Always a reaction passes through an energy
maxima (energy of activation) before product
formation. Enery of Activation is the energy
difference between the reactant and transition
state or activated complex.

17. Mechanism of action…
1) Energy changes during the reaction
a) Free energy of activation (EA): molecules
must have enough energy to surmount the
energy barrier. Uncatalyzed reactions have
high EA and hence are slow.
b) Rate of reaction: The lower the EA, the more
molecules will have sufficient energy to
surmount the energy barrier, the fastest the
rate of reaction. Enzymes lower the EA
hence hasten the rate of reaction.
c) Alternate route: enzymes lead reaction
through short cut with lesser energy

18. MECHANISM OF ACTION…
2) Chemistry of active site: is a complex
molecular machine employing varieties of
mechanisms to convert substrate to product.
Some factors responsible for enzyme
efficiency include:
a) Transition state stabilization: by forming a
geometric structure similar to the substrate.
b) Other ways; acid base catalysis by accepting
proton or forming covalent enzyme-substrate
complex
c) Transition state can be studies using
spectroscopic techniques

19. Factors Affecting rate
• Enzymes can be isolated from cells and their
properties studied.
1) Substrate concentration: Rate of enzyme
catalyzed reaction increases with increase in [S]
until the maximal velocity (Vmax) is reached.
NB: after Vmax is reached increasing the [S] will not
have any effect on reaction rate due to saturation
(active sites are occupied by reactants forming
ES).
Hence enzymes follow saturation kinetics.

20. Factors affecting….
2) Temperature:
Increase in T increases reaction rate to reach
maximum activity due to increased number of
molecules having sufficient energy but further
increase lowers the rate due to denaturation of
the enzyme (loss of native conformation hence
loss of activity)
The T at which the enzyme is 100% active is
termed the optimum temperature.

21. Factors affecting …
3) pH: Same to T pH is required for enzyme
activity but further increase in pH due to
denaturation of enzyme brings in sharp decline
in enzyme activity hence reaction rate.
NB: The pH at which an enzyme is 100% active is
termed the optimum pH. The optimum pH varies
for different enzymes. Some are active at neutral
pH some at acidic pH and some at neutral pH.
4) Inhibitors also affect enzyme activity by
attaching to the enzyme and affecting it’s the
conformation at the active site.

23. Enzyme Kinetics
• The field of Biochemistry that studies the
quantitative measurement of the rates of
enzyme-catalyzed reactions and the systematic
study of factors that affect the rates of enzyme
catalyzed reactions.
• Kinetic analyses permit scientists to reconstruct
the number and order of the individual steps
by which enzymes transform substrates into
products.

24. Enzyme Kinetics…
• Changes in free energy determine the
direction & equilibrium state of chemical
reactions.
• The Gibbs free energy change ΔG (also called
the free energy or Gibbs energy) describes
both the direction in which a chemical reaction
will tend to proceed and the concentrations of
reactants and products that will be present at
equilibrium.
ΔG0 = − RT ln Keq

25. Enzyme Kinetics…
• Enzymes lower the Energy of Activation but do
not affect the equilibrium.
• In presence of the enzyme (E) the equilibrium
constant for a reaction,
A + B + Enz → P + Q + Enz
Keq = [ P] [Q] [Enz] = [P] [Q]
[A] [B] [Enz] [A][B]
NB: The enzyme has no effect on the Keq as it
cancels out in the equilibrium expression.

26. Enzyme Kinetics…
• Measuring rate is so difficult as concentration of
substrate [S] varies during the course of reaction.
• We assume steady state i.e. a state where the
enzyme-substrate complex concentration [ES] and
other intermediates becomes a constant over time.
• We measure initial velocity by taking different [S] each
time.
• At one point increase in [S] will not have any effect on
the rate because of saturation (all active sites on
enzyme) occupied by substrate. This velocity is
termed the maximum possible velocity (Vmax) of that
enzyme catalyzed reaction.
• Such kinetic study is termed steady-state kinetics.

27. Enzyme Kinetics…
• Consider the enzyme catalyzed reaction
E + S ES E + P
Vo =k2[ES]
where k2 is rate constant for 2nd reaction
Rate of ES formation =k1([Et] – [ES]) [S]
Rate of ES breakdown = k-1[ES] + k2 [ES]
Assuming initial rate reflects steady state hence rate of
formation and breakdown of ES are equal
k1([Et] – [ES]) [S] = k-1[ES] + k2 [ES]
k1
K-1
k2

28. Enzyme kinetics…
• Solving for ES and rearranging
k1[Et][S] - k1[ES][S] = (k-1 + k2)[ES]
Adding k1[Et][S] to both sides of eqn and simplifying
k1[ES][S] = (k1[S] + k-1 + k2 )[ES]
Solving this eqn for [ES]
[ES] = [Et][S] = [Et][S]
k1[S] + k-1 + k2 [S] + (k-1 + k2 )/k1

29. Enzyme kinetics…
Or simply [ES] = [Et][S]/Km + [S] substituting Vo interms
of [ES] we get
Vo = k2 [Et][S] or
Km + [S]
This last equation is termed the Michaelis-Menten
equation for enzyme catalyzed reactions
Vo = Vmax [S]
Km + [S]

30. Regulation of Enzyme Activity
1) Feedback inhibition: some enzymes are inhibited by
their own final product
E1 E2 E3
A ↔ B ↔ C ↔ D
Note here that the final product D inhibits the first
enzyme E1 and hence the whole reaction is slowed
down.
Eg: Conversion of L-threonine to L-Isoleucine where the
1st enzyme threonine dehydrogenase is inhibited by
the final producct Isoleucine

31. Regulation of Enzyme….
2) Allosteric regulation: activity of some enzymes
is regulated by molecules other than their
substrates called allosteric modifiers. Such
molecules alter the conformation of active sites
of allosteric enzymes.
Such enzymes have another site different from
the active site in their folded structure called the
allosteric site.
Eg. Phosphofructokinase is allosterically
regulated by ADP, AMP, ATP and Citrate. The
former inhibit it but the latter activate it.

32. Regulation of Enzyme…
3) Covalent modification: some enzymes change
their activities through a reversible covalent
modification of their structure.
Eg: Phosphorylation dephosphorylation of fatty
acid synthase and glycogen phosphorylase.
NB: Phosphorylated form of glycogen
phosphorylase is active but dephosphorylated
form of glycogen synthase is active.

33. Regulation of Enzyme…
4) Zymogen activation: Proteolytic enzymes are
produced in their inactive precursor form called
zymogens but after losing part of their peptide
they become active enzymes.
Eg: Pepsinogen, Chymotrypsinogen and
Trypsinogen are inactive zymogen forms of the
active enzymes Pepsin, Chymotrypsin and
Trypsin.Some proteins are also produced in
their inactive preprotein form like preinsulin.
Others are the blood clotting enzymes thrombin
and fibrin (Prothrombin, fibrinogen).

34. Regulation of enzyme…
5) Induction or Repression of Enzymes: Cells
can regulate the amount of enzyme present
by altering the rate of enzyme synthesis
The increased (induction) or decreased
(repression) of enzyme synthesis leads to
alteration in the total amount of active sites.
Induction and repression are slow processes
that may be elicited by hormone release. Eg.
Insulin increases the synthesis of key
glycolytic enzymes.

35. Measurement of Km and Vmax
• Km and Vmax values of an enzyme can be
determined from a plot of the initial velocity of
the reaction for a range of [S]
• However, because the velocity curve is
hyperbolic, it can be difficult to determine the
asymptote that defines Vmax and the halfway
point that determines Km.
• These days, a computer program can
extrapolate the curve that involves plotting the
reciprocal of the velocity against the reciprocal
of the substrate concentration, which gives a
linear curve instead of a hyperbolic one:

36. Lineweaver-Burk Plots
NB: We can get this equation by taking reciprocal
of the Michaelis-Menten equation.
Vo= Vmax [S]
Km + [S]
1 = Km + 1
Vo Vmax [S] Vmax

37. Inhibition of Enzymes
• Enzymes catalyze virtually all biological
reaction but molecular agents called inhibitors
can interfere and slow or halt enzymatic
reactions.
• Two broad catagories of inhibition
1) Reversible inhibition
2) Irreversible inhibition

38. Reversible Inhibition
• Are of three types:
1) Competitive inhibition: inhibitors [I] that compete
with substrate for the same active site on enzyme.
Such inhibitors have structure similarity with substrate.
Attachment of the inhibitor is reversible and hence the
effect can be abolished my increasing [S]. When [S]
increases the effect of the I in competing for active site
is very much minimized.
Hence the effect of competitive inhibitor is to increase
the Km without affecting the Vmax .
Km decreased Vmax unchanged

39. Classical Competitive Inhibition

40. Noncompetitive Inhibition
• In noncompetitive inhibition, the substrate and
the inhibitor can both be bound to the enzyme
at the same time, but the bound inhibitor
renders the enzyme inactive. The inhibitor binds
at a site different from the active site.
• This reduces the effective Vmax, but since
substrate binding is not affected by the inhibitor,
the Km value remains the same

41. Noncompetitive inhibition

42. Uncompetitive Inhibition
• In general, the effect of inhibitors can be more
complex, with both Km and Vmax being affected.
Such cases are referred to as mixed inhibition.
• Uncompetitive inhibitor binds at a site distinct
from the substrate active site and unlike
competitive inhibitor binds to only the ES
complex.
• Mixed inhibitor will bind to distinct site but will
bind to both the E or ES .
• Uncompetitive inhibitors alter both Vmax and Km
Hence both are decreased in this case

43. Uncompetitive Inhibition
Uncompetitive mechanism model

44. Uncompetitive Inhibition
• Note both Km and Vmax changing

45. Irreversible Inhibition
• Inhibitors are those that cobine with or destroy
a functional group on an enzyme essential for
the enzyme’s activity.
• Useful in the study of reaction mechanisms.
• Special class of irreversible inhibitors are called
suicide inactivators.
• Poisons like warfarin act as irreversible inhibitor
of prothrombin (useful in blood clotting)
formation and causes death by internal
bleeding

46. Application of Enzyme Inhibition
• This principle is applied in drug synthesis and at
least half of modern drugs act as enzyme
inhibitors.
• Competitive inhibition is applied in the treatment
of gaut. The drug allopurinol is structurally
similar to hypoxanthine, which is a substrate of
the enzyme Xanthine oxidase.
• Gaut is caused due to increased oxidation of
purine bases that produces uric acid, which
forms an insoluble sodium-salt that precipitates
between joints and causes inflamation.

47. Application of Enzyme Inhibition…
• The β-lactam drugs like penicilline and
amoxicilline act by inhibiting enzymes involved
in bacterial cell wall synthesis.
• Angiotensin-converting enzyme inhibitors like
captopril, enalapril and lisinopril lower blood
pressure by blocking the enzyme that cleaves
angiotensin I to form the potent vasoconstrictor,
angiotensin II.
• Competitive inhibitors like statin drugs Levastatin ,
atorvastatin (Liptor), simvatatin (Zocor) inhibit
HMGCoA reductase the enzyme catalyzing the
commited step in cholesterol synthesis

48. Application of Enzyme Inhibition
• Noncompetitive inhibitors like Pb (heavy metal)
inhibits the enzyme ferrocheletase that chelates
Fe2+ to protoporphyrin to form heme. The Pb
attaches competitively to the sulfhydryl residues
on the enzyme protein and inhibits the enzyme.
• Some insecticides that have a neurotoxic effect
irreversibly bind to the active site of the enzyme
acetylcholinesterase (an enzyme that cleaves
the neurotransmitter acetylcholine).
• Many HIV-AIDS drugs act as enzyme inhibitors
(integrase inhibitors, inhibitors of reverse
transcriptase, protease inhibitors, fussion
inhibitors etc)

49. Enzymes in Clinical Diagnosis
• Enzymes are useful in the diagnosis of
diseases.
• Eg. Aspartate transaminase and alanine
transaminase enzymes are used to diagnose
liver diseases
• Lactate dehydrogenase, creatine kinase are
used for diagnosis of heart diseases

50. Industrial application
• Enzymes are used for large scale
production in in food, pharmaceutical,
beverage, plastic, fertilizer etc industries