Enzymes are proteins that act as catalysts and speed up chemical reactions without being consumed. They are highly specific and function most effectively within a certain pH range and temperature. The active site of the enzyme binds to the substrate and the transition state requires lower activation energy than without the enzyme. The turnover number refers to the number of substrate molecules an enzyme can convert to products per unit time. Enzyme activity is affected by factors like substrate and inhibitor concentration, pH, temperature, and cofactors. Different types of inhibitors like competitive, non-competitive, and allosteric inhibitors can bind to enzymes and decrease their activity.

1. Enzymes
Prof Dr: Olfat Shaker
Department of Medical Biochemistry

2. Introduction
• Enzymes are Proteins but in
some cases they are RNA
e.g.snurps (ribozymes).
• They are Catalysts produce by
living cells, can work outside
these cells.
• They Speed reactions without
changing the equilibrium point.
• They denatured at high temp

3. • They function in minute amounts, remain
unchanged chemically during the reaction.
• Enzymes are highly specific.
• They are sensitive to changes in pH and
temperature.
• They lose some of their activity during the
reaction

4. Turnover number
The “turnover number” is the number of
substrate molecules converted into product by
an enzyme molecule in a unit time when the
enzyme is fully saturated.

5. Chemical nature of Enzymes
1. Simple proteins: only protein chain(s)
2. Conjugated proteins (Holoenzyme): consists of
Protein part (apoenzyme)
Non-protein part : may be
Organic (coenzymes): loosely attached to apoenzyme
Inorganic (activator): loosely attached to apoenzyme
*Prosthetic group is a non-protein part firmly attached to the apoprotein

6. Enzyme Terminology
• Hydrolyzing enzymes are named by adding –ase to the
name of substrate
– sucrase – reacts sucrose
– lipase - reacts lipid
• Enzymes named by adding their function to the name of
the substrate
– Lactate dehydrogenase – remove hydrogen from lactic acid
• Pepsin and trypsin

7. Localization of the enzyme
• Intracellular enzymes :they act inside the
cells that make them.
• Extracellular: they act outside the cells
that secret them such as:
• digestive enzymes in the GIT
• coagulation enzymes in plasma.

8. Zymogens
• Digestive and coagulation enzymes are
secreted in inactive forms, zymogens or
proenzyme.
• Zymogens are activated by trimming of a short
peptide blocking the active site, or by covalent
modification of the zymogen.

9. Structure of the enzyme
• The enzyme is tertiary or quaternary
structure that has spatial configuration.
• It has pockets on its surface. Each pocket
has its own function.
–The catalytic or active site.
–The allosteric site.

10. The catalytic or active site
It is the site at which the substrate
binds to the enzyme.
It should be fit to the substrate
(fitness is made by the tertiary
structure of the enzyme molecule).
Any factor affecting this structure
will alter the fitness and formation of
enzyme-substrate complex.

11. The Allosteric site
It is a site for fitting of a small molecule whose
binding alters the affinity of the catalytic site to the
substrate.
This small molecule is called allosteric modifier
stimulatory (making it more fit)
inhibitory (making the catalytic site unfit)
for binding of the substrate.

12. The Enzyme Action

13. Mode of enzyme action
• The reactants should raise their energy levels
to reach a transition state.
• The transition state represents the energy
barrier between the reactants and products.
• This energy is known as energy of activation.
• The enzyme makes the reaction needs lower
activation energy.

14. Activation Energy and Catalysis

15. Factors affecting enzyme action
• The activity of the enzyme is
evaluated by measuring the rate or
the velocity of the reaction.
• velocity of the reaction is measured
by how many moles of the substrate
are converted into products per unit
of time (minute).

16. Factors affecting Enzyme activity
• Substrate concentration
• Temperature
• pH
• Time
• Cofactors
• Enzyme Inhibitors

17. Substrate Concentration
As substrate
concentration increases,
• the rate of reaction
increases (at constant
enzyme concentration).
• the enzyme eventually
becomes saturated
giving maximum
activity.

18. Michaelis constant (Km)
• Km is equal to the substrate
concentration [S] at which the
reaction is half of its maximum
(½Vmax).
• It expresses the affinity of the
enzyme to its substrate.
• Low Km means high affinity of the
enzyme to the substrate
• High Km means low affinity of the
enzyme to the substrate

19. Enzymes – Lineweaver-Burk Equation
V = Vmax [S]
[S] + Km
Inverting the Equation yields: 1 = Km 1 + 1 .
(Lineweaver-Burke Equation) V Vmax [S] Vmax
By plotting 1/ V as a function of 1/[S],
a linear plot is obtained:
Slope = Km/Vmax
y-intercept = 1/Vmax

20. Michaelis-Menton Equation: Lineweaver-Burk Equation:
Comparison of these two methods of plotting the same data:

21. Temperature
Enzymes
• are most active at an
optimum temperature
(usually 37° in humans).
C
• show little activity at low
temperatures.
• lose activity at high
temperatures as
denaturation occurs.

22. pH
Enzymes
• are most active at
optimum pH.e.g:
• Pepsin is 2.0
• Trypsin is 6.0.
• Alkaline phosphatase is 9.5
• lose activity in low or
high pH as it undergoes
Denaturation.

23. Time
As time is passed the rate of the enzyme-
catalyzed reaction diminishes due to:
–Decline of substrate concentration
–The accumulated product may cause feed-
back inhibition of the enzyme

24. Cofactors
• Most enzymes are associated with non-protein part which may be:
metal-ion as zinc, copper, iron
prosthetic group as flavine nucleotide, biotin, 4’-
phophopantetheine (of pantothenic acid), or cobamide.
coenzyme as nicotinamide nucleotide, NAD or NADP
• All these cannot be isolated from the protein part of the enzyme (apo-
enzyme) by dialysis.
• The protein part of the enzyme (apo-enzyme) + [metal, prosthetic
group, or the coenzyme] = holoenzyme

25. Inhibitors (I)
Inhibition may be reversible or irreversible.
• Reversible: If the inhibitor binds non-covalently to
the enzyme
• Irreversible: If the inhibitor binds covalently to the
enzyme
If [S] is > [I], only part of enzyme molecules are
occupied by the [I] and the inhibition will be
proportionate to [I].
[I] increases Km, so, more substrate is needed to
reach Vmax.

27. Reversible Inhibitors
Competitive inhibitors: compete with the
substrate for the same active site (catalytic site)
Non-competitive inhibitors: bind to the enzyme
in a location other than the active site
Allosteric inhibitors: bind to the enzyme in the
allosteric site, produce conformational change
leading to enzyme inhibition.

28. Competitive inhibitors
• If [I] is > [S], E-I complex is formed, and it fails to dissociate.
So, inhibition of the E-S complex reaction takes place.
• Examples of competitive inhibitors:
• Allopurinol competes with hypoxanthine for xanthine oxidase
inhibiting the formation of uric acid, so it is used in treatment of
hyperuricemia (gout).
• Dicumarol or Warfarine compete with vitamin K, for epoxide
reductase, so they are used to reduce prothrombin synthesis.
• Statins (e.g. atorvastatin) competes with HMGCoA for its reductase,
so, it inhibits cholesterol synthesis.
• Methotrexate competes with dihydrofolic acid for dihydrofolate
reductase, so, it inhibits DNA synthesis and used in treatment of
cancers.

30. Non-competitive inhibitors
• The inhibitor binds to the enzyme in site away
from the catalytic site.
• Inhibition cannot be reverted by increasing [S].
• Km is not changed because the inhibitor does
not interfere with E-S formation, but Vmax is,
apparently reduced.

31. Allosteric inhibitors
• Allosteric inhibitors are low-molecular weight
substances that bind the allosteric sites, on the surface
of the enzyme.
• This interaction causes conformational changes in the
catalytic site that makes it unfavorable for binding to
substrate.
• The importance of allosteric inhibition is to avoid
accumulation of the final product of succession of
reactions (metabolic pathway).
• End- product of a metabolic pathway inhibits the initial
enzyme in the pathway, this called feed-back inhibition.

32. Feedback Inhibition

33. Irreversible enzyme Inhibitor
Examples:
Lead binds to ferrochelatase during heme synthesis.
Thiol group blocking agents, e.g.
Iodoacetate inhibits glyceradehyde-3-P
dehydrogenase
Heavy metal bridges two –SH groups making Enz-
S-Metal-S-Enz,
Oxidizing agents that oxidize –SH into disulfide (-S-
S-)
Protein precipitants as
alkaloidal reagents, e.g. tungestic acid
denturating agents, e.g. Heating, conc. acids or
alkalies.

34. Regulation of Enzyme activity
1. Covalent modification:(short term regulation)
The enzyme may be phosphorylated in the OH group of
serine, threonine, or tyrosine. This process is catalyzed by
kinase. ATP is the source of phosphate.
The phosphoprotein produced is dephosphorylated by
phosphatase.
phosphoprotein enzyme is the active form of the
enzyme, e.g. glycogen phosphorylase.
the dephosphorylated form is the active form of the
enzyme, e.g. glycogen synthase.

35. 2. Induction and repression of enzyme
synthesis
Enzymes synthesis (long term regulation ).
• Synthesis is stimulated by "induction" or inhibited by "repression".
• Control of enzyme synthesis is under hormonal control that affects gene
expression related to that particular enzyme.
• Example:
• Insulin induces synthesis of glucokinase and phosphofructokinase, but represses
glucose-6-phosphatase and fructose 1,6,bisphosphatase.
• Steroid and thyroid hormones effects on enzyme generation.
• Control of enzyme activity by induction or repression consumes hours or
days to be achieved.
3. Allosteric modification (short term regulation)
occurs within seconds or minutes.

38. Plasma enzymes
• The only functioning plasma enzymes are those of
blood coagulation secreted from the liver as
zymogens.
• The plasma enzymes are diffused from tissues into
plasma due to turnover of tissue cells or due to
diffusion of these enzymes out of the diseased cells
(those undergoing degenration or involved in
inflammation).
• Plasma enzymes lack tissue specificity, because the
same enzyme is mostly secreted from more than
one tissue type.

39. • Examples of plasma enzymes
– Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) are present in
RBCs, liver, myocardium, and skeletal muscle.
– Creatine kinase in present in muscle, heart, and brain.
• Their rise in plasma is not, by-itself, diagnostic to a disease.
They may be of value to follow-up a disease already
diagnosed.
• Isoenzymes (Isozymes)
• When the enzyme is a quaternary structure, some of its subunits are not
identical according to tissue of origin. However, all perform the same
catalytic function. They can be differentiate by electrophoresis.
• Examples:
• LDH has five isozymes
• CK has three isozymes
• alk.phosphatase has three isozymes.
• Isozymes, accordingly, move differently in electrophoresis field.

40. Thank you