g

1. Advanced enzymology
For PG-Medical Biochemistry
Students
Belay Zewdie (MSc, Assistant
professor)
Faculty of Medical Sciences
May,2023

2. Learning objectives
At the end of this sub-topic, the students will be
able to
 Over view of enzymes
 Describe the components of enzymes
 Know the structure, function of coenzymes in different
metabolic reactions
 Know the multi-enzymes system occurrence, isolation and
properties

3. Over View Summary of Enzymes
Enzymes
 Protein catalysts that increase the velocity of a chemical reaction
by lowering the energy of the transition state
 Are not consumed during the reaction they catalyze
 Molecules contain a special pocket or cleft called the active site
 Contains amino acid side chains that participate in substrate
binding and catalysis
 binds the substrate, forming ES complex
– Binding cause a conformational change in the enzyme
(induced fit) that allows catalysis
– ES is converted to EP, which subsequently dissociates to
enzyme and product
 Allows a reaction to proceed rapidly under conditions prevailing
in the cell by
 providing an alternate reaction pathway with a lower free
energy of activation

4.  Does not change the free energies of the reactants or products
therefore, does not change the equilibrium of the reaction
 Most enzymes show Michaelis-Menten kinetics, and a plot of
the initial reaction velocity (vo) against substrate concentration
([S])
has a hyperbolic shape similar to the oxygen dissociation
curve of myoglobin
 Any substance that can diminish the velocity of such enzyme-
catalyzed reactions-Inhibitors
 The two most commonly encountered types of reversible
inhibition are
competitive (which increases the apparent Km) and
noncompetitive (which decreases the apparent Vmax)
Over View Summary of Enzymes

5. Over View Summary of Enzymes
 In contrast, the multi subunit allosteric enzymes frequently
show
a sigmoidal curve similar in shape to the oxygen dissociation
curve of hemoglobin
They typically catalyze the committed step (often the rate-
limiting or slowest step) of a pathway
they regulated by molecules called effectors (also modifiers)
that bind noncovalently at a site other than the active site
Effectors can be either positive (accelerate the enzyme-
catalyzed reaction) or negative (slow down the reaction).
Effector can alter the affinity of the enzyme for its
substrate, or modify the maximal catalytic activity of the
enzyme, or both
 Can also be regulated by covalent modification
 Changes in the rate of synthesis or degradation
 Have diagnostic and therapeutic value in medicine

6. Over View Summary of Enzymes

7. Over View Summary of Enzymes

8.  Almost all the enzymes are proteins having well
defined structures.
 Some functional groups are close enough in
space to form a portion called the active center.
 Active centers look like a cleft or a crevice.
 Active centers are hydrophobic.
Active Center

9. Active Center

10. Lysozyme
Residues (colored ) in the active site come from
different parts of the polypeptide chain .

11.  The active center has two essential groups in general
1. The catalytic site
 the region that catalyzes the chemical reaction
 the site(s) which manipulates the substrate to help
reaching the reaction TS and equilibrium faster.
 It may be slightly separated from the substrate-
binding site or they may be integrated into one site.
2. The substrate-binding site
 the site at which substrate specifically binds and
activates the chemical action-along with the
catalytic site.
Active Center

12. +
- Catalytic group
Binding group
Substrate
molecule
Protein chain
Active center
Essential groups
outside the
active center
Active Center

13. Active centers

14. Allosteric Site
The allosteric site
 additional binding site that does not have a catalytic
function but
o has a regulatory function on
• the enzyme substrate binding and/or
• catalytic functions
• The term allosteric site means
– “the other steering site”, i.e., other than and
separated from the catalytic/substrate-binding
site(s); and
– allostery means “a change in shape”

15. +
Substrate
Active site
Allosteric Site
+
+ + No binding due to
lowered substrate
affinity
Allosteric enzyme
Allosteric
activator site
Allosteric
inhibitor site
Allosteric
effector
Binding
due to
increased
substrate
affinity

16.  Simple enzymes: consists of only one
peptide chain
 Conjugated enzymes:
holoenzyme = apoenzyme + cofactor
(protein) (non-protein)
 Cofactors:
• metal ions
• small organic molecules
Molecular Components

17. • Simple enzymes like trypsin, pepsin, and
urease
• do not require cofactors,
• however other enzymes do require small
non-protein molecules called cofactors,
• and these enzymes are referred to as
conjugate enzymes.

18. Molecular Components

19. Cofactors
 Groups that contribute to the reactivity of enzymes
beside amino acid residues
 These groups are called cofactors
 chemicals required by apoenzymes
(inactive)
 to become holoenzymes (active)
 There are two types of cofactors
1. Essential ions - metal ions –inorganic
2. Coenzymes - organic molecules that act as group-
transfer reagents (accept or donate groups)- can also
be H+ and/or e-

20. Cofactors
A coenzyme
 a non-protein organic substance which is
dialyzable, thermostable and loosely attached to
the protein part.
A prosthetic group
 an organic substance which is dialyzable and
thermostable which is firmly attached to the
protein or apoenzyme portion.
 A metal-ion activator
 these include K+, Fe++,Fe+++,Cu++,Co++,Zn++,Mn++
,Mg++,Ca++

21. Metal ions
• Metal-activated enzyme
– ions necessary but loosely bound.
– Often found in metal-activated enzyme.
• Metalloenzymes
– Ions tightly bound.
Particularly in the active center
Transfer electrons
Bridge the enzyme and substrates
Stabilize enzyme conformation
Neutralize the anions.

22.  Loosely bind to apoenzyme. Be able to be
separated with dialysis.
 Accepting H+ or group and leaving to transfer it
to others, or vise versa.
Coenzymes
Prosthetic groups
 Tightly bind through either covalent or many
non-covalent interactions.
 Remained bound to the apoenzyme during
the course of reaction.

23. Coenzymes
 Coenzymes
complex that participate in catalysis by providing
 functional groups
 In humans, they are usually synthesized from
vitamins
 Each coenzyme is involved in catalyzing a specific
type of reaction
Q1.Although coenzymes look like they should be
able to catalyze reactions autonomously, they have
almost no catalytic power when not bound to the
enzyme. Why?

24. • In order for a substrate to react with a coenzyme,
• it must collide with a coenzyme at exactly the right
angle.
• In addition to providing this proximity and
orientation, enzymes contribute in other ways,
• such as activating the coenzyme by extracting a
proton (e.g., TPP and CoA) or
• polarizing the substrate to make it more susceptible to
nucleophilic attack.

25.  Coenzymes can be divided into two general classes
1. Activation-transfer Coenzymes and
2. Oxidation-reduction Coenzymes
1. Activation-transfer Coenzymes
 participate directly in catalysis by forming covalent
bond of the Substrate
Coenzymes---

26. A. Thiamine pyrophosphate(TPP)
 used for breaking carbon-carbon bonds
B. Biotin
 activates and transfers CO2 to compounds in carboxylation
reactions
 Biotin is covalently attached to a lysine residue in the
carboxylase enzyme
C. Coenzyme A (CoA or CoASH)
 are synthesized from the vitamin pantothenate
(pantothenic acid)
 The active sulfhydryl group, binds to acyl groups (e.g.,
acetyl, succinyl, or fatty acyl) to form thioesters
D. Pyridoxal phosphate
 The functional group is a reactive aldehyde that forms a
covalent intermediate with amino groups of amino acids
(a Schiff base)
Coenzymes---

27. Activation-transfer coenzymes common features
1. a specific chemical group involved in binding to
the enzyme
2. a separate and differentiate functional or
reactive group participates directly in the
catalysis of one type of reaction by
 Forming a covalent bond with the substrate and
3. dependence on the enzyme for additional
specificity of substrate and additional catalytic
power
Coenzymes

29. Vitamins Used as Co-enzymes

30. 2. Oxidation-Reduction Coenzymes
 involved in oxidation-reduction reactions
catalyzed by enzymes categorized as
oxidoreductases.
 nicotinamide adenine dinucleotide (NAD+) and
flavin adenine dinucleotide (FAD)
 can transfer electrons together with hydrogen and
have unique roles in the generation of ATP from the
oxidation of fuels
 Vitamin E and vitamin C (ascorbic acid) are
oxidation-reduction coenzymes that can act as
antioxidants and protect against oxygen free
radical injury
Coenzymes---

31. Definition of multi-enzymes
• Multienzyme
– A protein possessing more than one catalytic function
contributed by distinct parts of a polypeptide chain
('domains'), or by distinct subunits, or both.
• Multienzyme complex
– A multienzyme with catalytic domains on more than
one type of polypeptide chain.
• Multienzyme polypeptide.
– A polypeptide chain containing at least two types of
catalytic domains.
• Catalytic domain.
– Any part of a polypeptide chain that possesses a
catalytic function. It may contain more than one
structural domain?????

32. Multi-enzyme system occurrence
 A Soluble and dissociated multi enzyme system
– With diffusing intermediates
 Multienzymes complex
– Intermediates in the reaction sequence catalyzd by
• enzyme complex are covalently and
• do not diffuse away from the complex
 Membrane bound enzyme system
– Large superamolar structure such as membranes
and ribozymes
– Succinate dehydrogenmnase
– aminopeptidase

33. Multi enzymes occurrence

34. • Each component of enzymes has an own
– km for substrate
– cofactors
–Vmax
–PH optimum
Multienzyme system Properties

35. Isolation, Purification And
Characterisation of Enzymes
1) Cell disruption(which can done via a number of
number of different processes of choice )
 Detergents lysis
 Osmolysis
 Freeze-thaw cycles
 Enzymatic lysis
 Ultrasonication
 Homogenisation
2) Centrifugation (at a specific speed depending on
the organ, tissue, organelle or fluid).
3) Removal of supernatant (Decantation to obtain
supernatant)

36. Methods for purification of enzymes
1. Centrifugation
2. Ion exchange
3. Gel permeation chromatography
4. Electrophoresis
5. Affinity chromatography,
6. Immunochemical techniques

37. Method of production /isolation of enzymes

38. Mechanisms of regulation
Metabolite channelling
 Channelling involves the metabolite shared by
two consecutive enzymes in a pathway may be
directly transferred from one to the other, without
being released into free solution, or
 at least without achieving equilibrium with the
metabolite in free solution

39. Metabolite channelling

40. Interconvertible enzyme cascades
• Cooperativity of interactions with individual
enzymes is an important way of making
feedback inhibition more effective as a
regulatory mechanism

41. Interconvertible enzyme cascade

42. The metabolic role of adenylate kinase
ATP + AMP = 2ADP
Effect of adenylate kinase.
(a)If the three adenine nucleotides are always in
equilibrium
 small changes in ATP concentration around 4 mM
result in large relative changes in the AMP
concentration.
(b)This allows an enzyme that binds AMP tightly
to show a much larger response to ATP than
it would have if there were no variations in AMP
concentration.

43. The metabolic role of adenylate kinase

44. The metabolic role of adenylate kinase

45. Reading Assignment
1. Adenylate Kinase and AMP Signaling
Networks: Metabolic Monitoring, Signal
Communication and Body Energy Sensing
Adenylate Kinase and AMP Signaling Networks:
Metabolic Monitoring, Signal Communication
and Body Energy Sensing - PMC (nih.gov)