enzimas, mecanismo de acción y clasificación

1. ENZYMES
STUDENTS:
MIGUEL AMAURY SALAS GARCÍA
KAREN PESQUEDA
ANA SILVIA FLORES

2. EXPECTED OUTCOMES
¡ Know the classification of enzymes
¡ Identifiy the specific characteristics of each of the enzyme groups.
¡ Understand the concept of co-enzyme and cofactor
¡ Comprehend the mechanism of action of the enzymes

3. CHARACTERISTICS OF ENZYMES
Almost all
enzymes are
proteins
Heat labile
Water soluble
Increase reaction
rate by lowering
activation energy
High specificity of
enzymes for
substrates
Do NOT alter
equilibria
NO permanent
changes in
enzymes occurs
during reactions
16% of their
weight as
nitrogen
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

4. CLASSIFICATION OF ENZYMES
Trivial names
Were given by adding the suffix “-ase” to the substrate.
IUBMB system of classification
¡ Complex but unambiguous.
¡ Name starts with EC (enzyme classification)
¡ Followed by 4 digits
Lactase
Wich acts in the
substrate lactose
Producing glucose +
galactose
First:
represents
the class
Second:
stands for
the subclass
Third: is the
sub-sub
class or
subgroup
Fourth: the
number of
particular
enzyme in
the list
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

5. Enzymes are grouped into
six mayor classes
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

6. CLASS 1. OXIDOREDUCTASES
This group catalyzes oxidation of one substrate with simultaneous reduction of another
substrate or co-enzyme.
Example:
Alcohol + NAD+ → Aldehyde + NADH + H+
Alcohol dehydrogenase or Alcohol-NAD-oxidoreductase or EC.1.1.1.1
AH2 + B A + BH2
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

7. ¡ Subclasses
Dehydrogenases
(hydride transfer)
Oxidases
(electron transfer
to molecular O2)
Oxygenases
(oxygen transfer
from 02)
Reductases
Peroxidases
(electron transfer
to peroxide)
Catalases Hydroxylases
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

8. CLASS 2.TRANSFERASES
This group transfers one group (other than hydrogen) from the substrate to another
substrate.
Example:
¡ Hexose + ATP → Hexose-6-phosphate + ADP
¡ Hexokinase or ATP-Hexose-6-phosphate-transferase
A-R + B A + B-R
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

9. ¡ Subclasses
Transaldolases Acyltransferases Glycosyltransferases Methyltransferases
Transaminases Kinases Phosphomutases
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

10. CLASS 3. HYDROLASES
This group hydrolyzes ester, ether, peptide or glycosidic bonds by adding water and then
breaking the bond.
¡ Example:
Acetylcholine + H2O → Choline + acetate
Acetylcholine esterase or acetylcholine hydrolase
Esterases Glycosidades Lipases
Proteases Nucleosidases
Bhatia S. Introduction to enzymes and their applications. Introd to Pharm Biotechnol. 2018;2(October).

11. CLASS 4. LYASES
This group can remove groups from substrates or break
bonds by mechanisms other than hydrolysis.
¡ Example:
Fructose-1,6-biphosphate → Glyceraldehyde-3-phosphate +
DHAP
Aldolase
Aldolases Decarboxylases Dehydratases Synthases
Some
pectinases
Bhatia S. Introduction to enzymes and their applications. Introd to Pharm Biotechnol. 2018;2(October).

13. CLASS 5. ISOMERASES
This group produces optical, geometrical or
positional isomers of substrates.
¡ Example:
Glyceraldehyde-3-phosphate → Dihydroxy acetone
phosphate
Triose phosphate isomerase
Epimerases Racemases
cis-trans
isomerases
Intramolecular
transferases
Bhatia S. Introduction to enzymes and their applications. Introd to Pharm Biotechnol. 2018;2(October).

14. CLASS 6. LIGASES
This group links two substrates together (usually
with hydrolysis of ATP).
¡ Example:
Acetyl CoA + CO2 + ATP → Malonyl CoA +
ADP + Pi
Acetyl CoA carboxylase
Synthetases
Bhatia S. Introduction to enzymes and their applications. Introd to Pharm Biotechnol. 2018;2(October).

15. TRANSLOCASES
This group assists in moving another molecule, usually across a cell membrane. Catalyze
the movement of ions/molecules across membranes or their separations with
membranes.
¡ Example:
“Side 1” “Side 2”
Carnitine-acylcarinitne
Transport carnitine-FA
complex
Across the inner
mithocondrial membrane
Bhatia S. Introduction to enzymes and their applications. Introd to Pharm Biotechnol. 2018;2(October).

16. CO-ENZYMES OR CO-FACTORS
¡ Consists of the non-protein part of the enzyme
¡ Prosthetic group or co-enzyme: organic molecules
¡ Divided into two groups
¡ Heat stable
¡ Metal cations: Co-factors
¡ The protein part of the enzyme
¡ It’s called the apo-enzyme
¡ Heat labile
Co-enzyme + Apo-enzyme = Holo-enzyme
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

17. ¡ FIRST GROUP OF CO-ENZYMES
The change occurring in the substrate is counter-balanced by co-enzymes.Thus, co-
enzymes may be considered co-substrates.
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

18. ¡ Nicotinamide Adenine Dinucleotide (NAD+)
¡ Co-enzyme synthesized from nicotinamide
Other examples: NADP-NADPH, FAD-FADH2 and FMN-FMNH2
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

19. ¡ Second group of co-enzymes
Participate in reactions transferring groups other than hydrogen.
¡ Example:
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

20. CO-ENZYMES ASVITAMIN
DERIVATIVES
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

21. ¡ Metallo-enzymes
Enzymes that require certain metals ions for
their activity.
¡ Example:
Copper in tyrosinase, the metal is tightly
bound with the enzyme.
Robinson PK. Enzymes : principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

22. MECHANISM OF ACTION OF ENZYMES
¡ Fundamental role of enzymes:
Accelerate biological reactions on specific substrates that will be transformed into
products.
Substrate
union
Catalytic
step
Enzymes
affect
reaction
rates, NOT
equilibria
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

23. ¡ Transformation of substrate into product it’s not immediate
Substrate
interacts with
active site
It’s converted
into a state of
transition
Residues of
union and
catalytic residues
Product
Very unstable, must be
stabilized by a’a in active site
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

24. LOWERING OF ACTIVATION ENERGY
¡ Defined:
Energy required
Convert all
molecules of a
reacting substance
From the ground
state to the
transition state
Substrates are
remaining in an
energy ground
Need to be
placed at a higher
energy level
So degradation
can occur
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

25. VELOCITY OF A REACTION IS RELATED TO ENERGY ACTIVATION
¡ Enzymes accelerate chemical reactions by creating
alternate pathways of lower activation energy
trough:
¡ Both situations occur thanks to the formation of E-S
complex, which also gives specificity.
Formation of covalent
bonds or transference of
functional groups
between enzyme and
substrate
Creation of non covalent
bonds between enzyme
and substrate with the
release of free energy
(union energy)
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

26. ¡ Numerous reactions produce temporary interactions between enzyme and substrate
that contribute to reduction of activation energy.
¡ Specifically accelerating catalysis reactions
¡ According to the mechanism of reduction for energy activation we can divide them
in 3 groups:
General acid-
base catalysis
Covalent
catalysis
Metalic ions
catalysis
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

27. ¡ General acid-base catalysis
A form of stabilizing the electric charges that appear in a state of transition is through
the transference of protons from or to the substrate.
¡ Proteases → performed by imidazol group of His.
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

28. ¡ Covalent catalysis
Several enzymes form covalent bonds between the substrate and the catalytic group of
the active site.
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

29. ¡ Metal ions catalysis
¡ Acting trough different mechanisms:
Stabilizing electro
charges in
transition state
Favoring
oxidoreduction
reactions
Modifying polarity
of certain bonds

30. ENZYME-SUBSTRATE INTERACTION MODELS

31. MICHAELIS-MENTEN THEORY
Enzyme-Substrate complex theory
(1913)
E + S ↔ E–S → E + P
enzyme (E)
substrate (S)
enzyme-substrate complex (ES)
product (P)
FISCHER'S TEMPLATE THEORY
Substrate fits on the enzyme, similar
to lock and key
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

32. KOSHLAND'S INDUCED FITTHEORY
Conformational changes are
occurring at the active site of
enzymes with the combination of
enzyme with the substrate.
A simplified explanation is that a
glove is put on a hand.
Substrate analog → some
structural alteration may
occur → reaction does
not take place → lack of
proper alignment
Allosteric inhibition
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

33. ACTIVE SITE OR ACTIVE CENTER OF ENZYME
Occupies only a small
portion of the whole
enzyme, in a crevice
The specific substrate is
bound, alignment of
specific groups or atoms
Conformational
orientation so as to
promote exact fitting
Bind by noncovalent
bonds, (hydrophobic
forces, hydrogen bonds).
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

34. ACTIVE SITE OR ACTIVE CENTER OF ENZYME
Catalytic residues or catalytic
groups: participate in making or
breaking the bonds
Substrate binding site and
catalytic site: may be separate.
Sreekumari, S;Vaidyanathan K.Textbook of Biochemistry for Medical Students. Seventh Ed. JAYPEE; 2013.

35. ENZYME KINETICS Application
Analysis, diagnosis, and
treatment of the enzymic
imbalances.
Targets of choice for drugs
Enzyme kinetics
Quantitative measurement
of the rates of enzyme-
catalyzed reactions
Systematic study of factors
that affect these rates
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

36. CHEMICAL REACTIONS ARE DESCRIBED USING
BALANCED EQUATIONS
• The double arrows
indicate reversibility
• Products: reactants
whose formation is
termodynamically
favored
• Functionally
irreversible under
physiologic
conditions.
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

37. CHANGES IN FREE ENERGY
Gibbs free energy change
ΔG
• Direction in which a
chemical reaction will
tend to proceed
• Concentrations of
reactants and products
that will be present at
equilibrium
• ΔGp minus ΔGs.
ΔG negative
•ΔGp < ΔGs
•The reaction is favored
(direction left to
right)→exergonic
•At equilibrium products
> substrates
•Keq >1
ΔG positive
•ΔGp > ΔGs
•The formation of
substrates will be
favored→endergonic
•Keq <1
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

38. THERMODYNAMIC
Reaction Description
Exergonic or
Exothermic
Reaction
Energy is released
Such reactions are
generally irreversible.
Isothermic
Reaction
Energy exchange is negligible
The reaction is easily
reversible
Endergonic or
Endothermic
Reaction
Energy is consumed and
external energy is to be
supplied.
This is usually accomplished
by coupling whit the
exergonic reaction
Is not possible to infer
from the overall ∆G
the number or type of
transition states through
which the reaction
proceeds
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

39. THERMODYNAMIC
¿Qué tipo de reacción es?
¿Qué tipo de reacción es?
¿Qué tipo de reacción
global es?

40. REACTION RATE AND ACTIVATION ENERGY
kinetic theory/
collision theory
Sufficient kinetic
energy for
reaching the
transition state
Collide “chocar”:
temperatura,
reactant
concentration
DGF Defines the Activation
Energy
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

41. REACTANT CONCENTRATION
The number of collisions will be proportionate to the
concentration of A and B
when n molecules of Areact
with m molecules of B
Rate constant, k
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

42. KEQ IS A RATIO OF RATE CONSTANTS
At equilibrium the overall
concentrations of
reactants and products
remain constant.
The rate of conversion of
substrates to products
therefore equals the rate
at which products are
converted to substrates
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

43. ENZYMES DO NOT AFFECT KEQ
All enzymes accelerate
reaction rates by
lowering ΔGF for the
formation of transition
states.
The presence of an
enzyme therefore has
no effect on ΔG0 for
the overall reaction
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

44. ASSAYS OF ENZYME CATALYZED REACTIONS
Measurements of the
rates of enzyme-
catalyzed reactions
Initial rate conditions
(traces of product
accumulate)
Initial velocity (Vi)
of the reaction is of
the rate of the
forward reaction.
As substrate
concentration is
increased,Vi increases
until it reaches a
maximum value
Vmax
The enzyme is said to
be “saturated”
with the substrate
Estimate the quantity
of enzyme present in
a biologic sample
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

45. Vi depends of [S]
Vi depends of
rapidity with
which product
dissociates from
the enzyme
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

46. THE MICHAELIS-MENTEN EQUATION
Relationship
between vi and [S]
The Michaelis
constant Km
The substrate
concentration at
whichVi is half the
maximal velocity
(Vmax/2) attainable
at a particular
concentration of
the enzyme
A.Vi= [S] C.Vi=Vmax B.Vi=Vmax/2
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

47. LINEAR FORM OF THE MICHAELIS-MENTEN EQUATION
Vmax y Km
Direct measurement
High concentrations
of substrate
A Linear Form of the
Michaelis-Menten
Equation (Vi y [S])
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

48. PARAMETERS TO COMPARE THE ACTIVITY OF DIFFERENT ENZYMES
Specific activity
Vmax divided by the
protein concentration
Turnover number
Vmax divided by the
moles of enzyme
present
Catalytic constant
kcat
Vmax divided by the
number of active sites
(St)
Catalytic efficiency
Ratio of two kinetic
constants: kcat and Km.
¿Para una mayor eficiencia
catalítica, cómo esperamos
que sea la Kcat y la Km
(altas o bajas) y porqué?
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

49. CATALYTIC EFFICIENCY, KCAT/KM
¿Qué
enzima tiene
mayor
eficiencia
catalítica?

50. CATALYTIC EFFICIENCY, KCAT/KM
¿Para cuál
sustrato
tiene mayor
eficiencia
catalítica la
fumarasa?

51. KDY KM
Dissociation
constant (Kd) for
complex ES:
Affinity of an
enzyme for its
substrate is the
inverse of Kd
Km often
approximates Kd
(k–1 ≫ k2)
Rodwell VW, et al. Harper's Illustrated Biochemistry. 30 ed. New York:McGraw-Hill, 2015.

52. FACTORS
INFLUENCING ENZYME
ACTIVITY

53. Enzyme Concentration
Effect of Substrate
Concentration
Velocity of reaction is increased
proportionately with the
concentration of enzyme.
As substrate concentration is increased,
the velocity is also correspondingly
increased in the initial phases; but the
curve flattens afterward.
Vmax

54. Michaelis Constant
Km: is substrate concentration (expressed in moles/L)
at half-maximal velocity.
It denotes that 50% of enzyme molecules are bound
with substrate molecules at that particular substrate
concentration.
Which enzyme has the highest
affinity for glucose: hexokinase or
glucokinase? Based on your answer,
what can we deduce about the km?
Hexoquinasa (km
0.05 mmol/L)
Glucosinasa
(10mmol/L)

55. Cooperative Binding
Enzyme has many subunits, and binding of
substrate to one unit enhances the affinity for
binding to other subunits.
Hill equation, originally described for explaining
the oxygen binding to Hemoglobin, is employed
(Hill was awarded Nobel prize in 1922).

56. Effect of Concentration
of Products
In a reversible reaction (S P) when
equilibrium is reached, as per the law of mass
action, the reaction rate is slowed down.
In inborn errors of metabolism, one
enzyme of a metabolic pathway is
blocked.
Enfermedad de la orina con
olor a jarabe de arce
Autosómico recesivo
Deficiencia de 2-cetoácido
deshidrogenasa de cadena
ramificada
leucina, isoleucina y valina
Rechazo alimentario, letargo,
vómitos, encefalopatía, cetosis

57. Effect of Temperature
The velocity of enzyme reaction increases
when temperature of the medium is increased;
reaches a maximum and then falls: Bell
shaped curve. Optimum
temperatu
re

58. Effect of pH
Each enzyme has an optimum pH, on both sides
of which the velocity will be drastically reduced.
Optimum pH may vary depending on the
temperature, concentration of substrate,
presence of ions, etc.
pH: 6-8

59. Enzyme Activation
In presence of certain inorganic ions,
some enzymes show higher activity.
Chloride ions activate salivary
amylase
Conversion of an inactive pro-enzyme or
zymogen to the active enzyme.
Trypsinigen
Trypsin

60. Enzyme Inhibition
Competitive
Inhibition
▪ Inhibitor molecules are competing with
the normal substrate.
▪ The inhibitor will be a structural analog
of the substrate.
▪ Competitive inhibition is usually
reversible. Or, excess substrate
abolishes the inhibition.
▪ The Km is increased in presence of
competitive inhibitor.
¿Cuál es la relevancia
clínica?

61. Non-competitive
Inhibition (Irreversible)
A variety of poisons, such as iodoacetate, heavy
metal ions (lead, mercury) and oxidizing agents
act as irreversible non-competitive inhibitors.
Increase in the substrate
concentration generally does not
relieve this inhibition.
Diisopropyl fluorophosphates
Serine of acetylcholinesterase.
Acetylcholine accumulatesand over-
stimulates autonomous nervous
system
Leads to vomiting, salivation, sweating,
and in worst cases even death
Activity can be regained only if new
enzyme is synthesized.

62. Enzyme Inhibition

63. Uncompetitive
Inhibition
Here inhibitor does not have any
affinity for free enzyme.
Inhibitor binds to enzyme–substrate
complex; but not to the free enzyme.
In such cases both Vmax and Km are
decreased.

64. Suicide Inhibition
The inhibitor makes use of the enzyme's own reaction
mechanism to inactivate it (mechanism based
inactivation).

65. Allosteric Regulation
Allosteric enzyme has one catalytic site where the substrate binds and another separate
allosteric site where the modifier binds.
Inhibit the activity of
the enzyme: allosteric
inhibition
POSITIVE
MODIFIER
Enhance the activity of
the enzyme: allosteric
activation
NEGATIVE
MODIFIER
POSITIVE OR NEGATIVE
COOPERATIVITY

66. ATP acts as an allosteric inhibitor (negative modifier) of PFK1.
Feedback Inhibition
Activity of the enzyme is inhibited by the final product of the biosynthetic pathway.

67. Induction / Repression
The inducer will relieve the
repression on the operator
site
Glucokinase is induced by insulin
Repressor acts at the gene
level
The effect is noticeable only
after a lag period of hours or
days

68. Covalent Modification
-Zymogen activation by partial proteolysis is an
example of covalent activation
-Protein
phosphorylation
The phosphate group may be
attached to serine, threonine or
tyrosine residues.

69. SPECIFICITY OF ENZYMES
Absolute Specificity: Urea is the only substrate for
urease
Bond Specificity: Trypsin can hydrolyze peptide bonds formed by carboxyl groups of arginine or lysine
residues in any protein.
Group Specificity: One enzyme can catalyze the same reaction on a group of structurally similar
compounds,
hexokinase can catalyze phosphorylation of glucose, galactose and mannose.

70. INTEGRATIVE SLIDE
ENZYMES
CLASSIFICATIO
N
MICHAELIS-
MENTENTHEORY
ENZYME
ACTIVITY
• Oxidoreductases
• Transferases
• Lyases
• Isomerases
• Ligases
Co-Enzymes
Mechanism of
action
Accelerate biological reactions on
specific substrates that will be
transformed into products.
Rol
Enzyme (E) combines with a substrate
(S) to form an enzyme- substrate (E-S)
complex, which breaks down to give
product (P)
Influenced by
Enzyme concentration,
substrate concentration, pH,
temperature and presence
of inhibitors.
• Competitive
• Non- competitive
• Uncompetitive
Allosteric
regulation

72. Objective:
Identify the effects of Germinated-Soy Milk (GSM) on Catalase (CAT) and Glutathione
Peroxidase (GSH-PX) activity in plasma, breast milk and BMI of breastfeeding mothers.
Subjects:
50
breastfeeding
mothers
0-6 months
breastfeeding
period
Age of 20-35
years old
Absence of
pathologies
Indonesia

73. Grouping research subjects and
intervention
Blood and breast milk samples
Group 1. (25
participants)
• GMS in daily diet (150
ml/day) for 2 months.
• Germitation of the soy
seeds overnight,
blended and served as
a drink.
Group 2. (25
participants)
• Placebo only (150
ml/day) for 2 months.
• Milk powder served as
a drink
0
1
2 months
Determination of CAT and GSH-PX activity
Body weight measured as BMI

74. Results
Effect of GSM on CAT and GSH-PX activity in plasma and breast milk.
Conclusions
GMS significantly increased activity of CAT an GHS-PX in plasma and breast milk. Therefore is suggested to be
consumed by breastfeeding mothers to provide suffciente amount of antioxidants in breast milk.