all about Enzymes 1234567891011121314.ppt

1. Enzyme

2. ENZYMES
Definitions--
A biomolecule either Protein or RNA, that catalyse a specific
chemical reaction, enhance the rate of a reaction by providing
a reaction path with a lower activation energy

3. The substances upon which an enzyme acts are traditionally called-
substrates
The selective qualities of an enzyme are collectively recognized-
specificity
The specific site on the enzyme where substrate binds and catalysis
occurs is called- active site

4. Fundamental Properties
1) Catalytic power-speeding up reactions 108 to 1020 fold.
They speed up reactions without being
used up.
2) Specificity
a) for substrate - ranges from absolute to relative
b) for reaction catalyzed
3) Regulated-- some enzymes can sense metabolic signals.

5. Active site
 Enzymes are composed of long
chains of amino acids that have
folded into a very specific three-
dimensional shape which contains
an active site.
 An active site is a region on the
surface of an enzyme to which
substrates will bind and catalyses a
chemical reaction.

6. e.g. H2O2
e.g. O2 + H2O
Progress of Reaction
Speeding up reactions

7. Mechanism of enzyme action
The enzymatic reactions takes place by binding of
the substrate with the active site of the enzyme
molecule by several weak bonds.
E + S ‹--------› ES --------› E + P
Formation of ES complex is the first step in the
enzyme catalyzed reaction then ES complex is
subsequently converted to product and free
enzyme.

8. "Lock and key" or Template model

9. Induced-fit model

10. Specificity
 Defined as the Selectivity of Enzymes for the Reactants Upon which They
Act
 In an enzyme-catalyzed reaction, none of the substrates is diverted into
nonproductive side reactions, so no wasteful by-products are produced.

11. Nomenclature / enzyme classification
 Trivial name (common name, recommended
name).
 Systemic name ( official name ).

12. Trivial name
1. Often named by adding the suffix -ase to the name of the
substrate upon which they acted
e.g. Urease, DNA Polymerase
2. Names bearing little resemblance to their activity
e.g. catalase, proteases

13. Systemic name
Each enzyme is characterized by a code no. called
Enzyme Code no. or EC number and contain four
Figure (digit) separated by a dot.
e.g. EC m. n. o. p
First digit represents the class;
Second digit stands for subclass ;
Third digit stands for the sub-sub class or subgroup;
Fourth digit gives the serial number of the particular
enzyme in the list.
e.g. EC 2.7.1.1 for hexokinase.

14. 2.7.1.1
ATP: glucose phosphotransferase
2- class name (transferase)
7- subclass name (phosphotransferase)
1- sub sub class (hydroxyl group as acceptor)
1- specific enzyme (D- glucose as phosphoryl group acceptor)

15. According to the IUB system of enzyme nomenclature
enzymes are grouped into 6 major classes
EC 1 OXIDOREDUCTASES
EC 2 TRANSFERASES
EC 3 HYDROLASES
EC 4 LYASES
EC 5 ISOMERASES
EC 6 LIGASES
IUB nomemclature

16. 1. Oxidoreductase
transfer of reducing equivalents from one redox system to another
e.g. Alcohol Dehydrogenase
Lactate dehydrogenase
cytochrome oxidase

17. 2. Transferase
functional group is transferred from one compound to another
e.g. kinases
transaminase
phosphorylase

18. 3. Hydrolase
cleave C-O, C-N, C-S or P-O etc bonds by adding water across the
bond
e.g.lipase
acid phosphatase
(important in digestive process)

19. 4. Lyases
cleave C-O, C-N, or C-S bonds but do so without addition of water and without
oxidizing or reducing the substrates
e.g. aldolase
fumarase
Carbonic anhydrase

20. 5. Isomerase
catalyze intramolecular rearrangements of functional groups that reversibly
interconvert to optical or geometric isomers
e.g. Triose isomerase
phosphohexose isomerase
mutase

21. 6. Ligase
catalyze biosynthetic reactions that form a covalent bond between two substrates
utilizing ATP-ADP interconversion
e.g. glutamine synthetase
DNA- ligase

22. Types of Specificity
 highly specific compared to other catalyst
 catalyzes only specific reaction
3 types
1. Stereospecificity/ optical specificity
2. Reaction specificity
3. Substrate specificity

23. Optical specificity
 able to recognize optical isomers of the substrate
 Act only on one isomer
e.g. enzymes of amino acid metabolism (D & L Amino acid oxidase)

24. Reaction Specificity
 catalyze only one specific reaction over substrate
e.g. amino acid can undergo deamination, transamination, decarboxylation and
each is catalysed by separate enzyme

25. Substrate specificity
specific towards their substrates
e.g. glucokinase and galactokinase- both transfer phophoryl group from ATP to different
molecule
3 types
a. Absolute
b. Relative substrate
c. broad

26. Absolute substrate specificity
 Act only on one substrate
e.g. urease

27. Relative substrate specificity
 act on structurally related substrates
 Further divide into
i. Group dependent- act on specific group e.g. trypsin- break peptide bond between
lysine and arginine, Chymotripsin act on aromatic AA
ii. Bond specificity- act on specific bond e.g. proteolytic enzyme, glycosidase

28. Broad specificity
 Act on closely related substrates
e.g. hexokinase- act on many hexoses

29. Chemical Nature & Properties of
Enzyme
 Protein or RNA
 Tertiary structure and specific conformation- essential for catalytic power
 Holoenzyme- functional unit
 Apoenzyme & coenzyme
Prosthetic group Coenzyme/cofactor
Non protein molecule Non protein molecule
Tightly (covalently)
bound
Loosely bound
Stable incorporation Dissociable
Cannot be dissociated Seperable by dialysis
etc

30.  Monomeric Enzyme- made of a single polypeptide e.g.
ribonuclease, trypsin
 Oligomeric Enzyme- more than one polypeptide e.g. LDH,
aspartate carbamoylase
 Multienzyme complex- specific sites to catalyse different
reactions in sequence. Only native conformation is active
not individual e.g. pyruvate dehydrogenase

31. Enzyme kinetics

32. 1. Enzyme concentration
2. Temperature
3. Hydrogen ion concentration or pH
4. Substrate concentration
5. Inhibitors
6. Product concentration
7. Activators
8. Physical agents
Factor affecting enzyme kinetics

33.  The rate of enzyme catalyzed reaction is directly
proportional to the concentration of enzyme.
 The plot of rate of catalysis versus enzyme concentrations
a straight line
Enzyme concentration

34.  Increase with temperature
 Bell shape curve
 Q10 (temperature coefficient)- factor by which the
rate of biological reaction increases for a 10ºC
increase in temperature
 Optimum temperature
 Mostly at body temperature
 Some enzyme may be active above body
temperature e.g. sanke venom phosphokinase,
muscle adenylate kinase, urease, enzymes in
thermophillic bacteria
Temperature

35. Effect of Temperature
Temperature(oC)
Reaction
Velocity
(v0)
 Rise or fall in enzyme activity with temperature is prominent survival feature in “Cold
blooded” animals
 In mammals- assumes physiological importance e.g. fever, hypothermia

36.  Bell shape curve
 Optimum pH
 Most show at neutral pH (6-8)
Since enzymes are proteins pH changes affect.
1. Charged state of catalytic site
2. Conformation of enzyme molecules
pH

37.  Trypsin- 7.6
 Pepsin- 2-2.5
 Acid phosphatase- 5
 Alkaline phosphatase- 9-10
 Enzymes from fungi- 4-6
Optimum pH for various enzyme

38.  Accumulation - decreases the velocity
 In biological system this is prevented by quick removal of product
Product concentration

39.  Inorganic metallic cation/anions acts as activators by combining with substrate,
ES complex, change in conformation of active site
 Metal activated enzymes- e.g. ATPase, Enolase
 Metalloenzyme- e.g.Pyruvate oxidase, cytochrome oxidase
Activators

40.  Make active site unavailable to substrate or
 Change enzyme structrure
Inhibitors

41.  Light, radiation ( u.v., X- rays, gamma rays etc)
e.g. salivary amylase- activity increased by red/ blue light whereas decreased by u.v.
light
Physical agents

42.  Rectangular hyperbola (Michaelis plot)
 Initial velocity- velocity when little substrate is reacted
Substrate concentration

43. Reasons for the three phases of the curve can be interpreted
1. In the first phase, substrate concentration is low and most of the
enzyme molecules are free so they combine with the substrate
molecules. Therefore, velocity is proportional to substrate
concentration. At this state, enzymatic reaction shows first-
order kinetics
2. In the second phase, half of the enzyme molecules are bound to
substrate, so the velocity is not proportional to substrate
concentration. At this stage, enzymatic reaction shows mixed-
order kinetics
3. In the third phase, all the enzyme molecules are bound to
substrate, so velocity remain unchanged because free enzyme is
not available though the substrate is in excess. At this stage
enzymatic reaction shows zero-order kinetics

44. A. Low [S] B. 50% [S] or Km C. High, saturating [S]

45.  The M-M equation was derived in part by making several assumptions. An
important one was: the concentration of substrate must be much greater
than the enzyme concentration.
 In the situation where [S] >> [E] and at initial velocity rates, it is assumed that
the changes in the concentration of the intermediate ES complex are very small
over time (vo).
 This condition is termed a steady-state rate, and is referred to as steady-state
kinetics. Therefore, it follows that the rate of ES formation will be equal
to the rate ES breakdown.
Steady State Assumption

46. Michaelis-Menten Equation Derivation
Rate of ES formation = k1([E][S] + k4([E][P]
=k1([ET] - [ES])[S]
(where [ET] is total concentration of enzyme E and k4 is considered neglible)
Rate of ES breakdown to product = k 2[ES] + k3[ES]
3
2 4
The enzyme is either present as free enzyme or as the ES complex
[E]total = [E] + [ES]

47.  Thus for the steady state assumption:
k1([ET] - [ES])[S] = k3[ES] + k2[ES]
k1([ET] - [ES])[S] = (k3 + k2)[ES]
Rearrange to define in terms of rate constants:
([ET] - [ES])[S] / [ES] = (k3 + k2) / k1
[ET] [S] - [ES][S] / [ES] = (k3 + k2) / k1
[ET] [S] / [ES] - [ES][S] / [ES] = (k3 + k2) / k1
([ ET] [S] / [ES]) - [S] = (k3 + k2) / k1
Define a new constant, Km = (k3 + k2) / k1
([ET] [S] / [ES]) - [S] = Km
Solve for the [ES] term (for reasons that will be given in the next step):

48. [ES] = [ET] [S] / (Km + [S])
The actual reaction velocity measured at any
given moment is given by:
V = k3[ES]
V = k3[ET] [S] / (Km + [S])

49. The maximum possible velocity (Vmax) occurs when
all the enzyme molecules are bound with substrate
[ES] = [E]total, thus:
Vmax = k3[E]total
Substituting this into the prior expression gives:
V = Vmax [S] / (Km + [S])
This is the mathematical expression that is
used to model your experimental kinetic data
It is known as the Michaelis-Menten equation

50.  
 
S
K
S
v
v
M
max


Michaelis-Menten Equation
In which:
v initial reaction velocity at [S]
KM the Michaelis constant
vmax the maximum possible initial reaction velocity

51. The substrate concentration that produces half the maximal velocity (Vmax/2) is
known as Michaelis constant (Km )
Michaelis Constant (Km )

52. Michaelis constants have been determined for many of the commonly used
enzymes. The size of Km tells us several things about a particular enzyme:
 A small Km indicates that the enzyme requires only a small amount of
substrate to become saturated. Hence, the maximum velocity is reached at
relatively low substrate concentrations.
 A large Km indicates the need for high substrate concentrations to achieve
maximum reaction velocity.
 The substrate with the lowest Km upon which the enzyme acts as a catalyst
is frequently assumed to be enzyme's natural substrate, though this is not
true for all enzymes.
 A Km of 10-7 M indicates that the substrate has a greater affinity for the
enzyme than if the Km is 10-5 M.
Meaninig of Km

53. 1. enzyme kinetic constant.
2. Indicates the substrate concentration required for the enzyme to work
efficiently
3. Low Km indicates high affinity of enzyme towards substrate. And vice-versa.
Hence,(Km α 1/affinity)
e.g. Hexokinase and glucokinase
Km of hexokinase is low (1 × 10–5 M) whereas Km of glucokinase is high (2.0 ×
10–2 M)
4. Km is required when enzymes are used as drugs
5. Use of enzymes in immunodiagnostics (ELISA) require Km of the enzyme
Significance of Km

54. Lineweaver-BurK Plot
V = reaction velocity (the reaction rate),
Km = Michaelis-Menten constant,
Vmax = maximum reaction velocity
[S] = the substrate concentration

55. Principal Ways of Regulating Enzymes
Competitive Inhibition
Allosteric Inhibition
Covalent Modification (phosphorylation)

56. -
HO
OH
HO
OH
+
HO
OH
HO
OH
Competitive
Inhibitors:
bind to active site
“unproductively”
and block
true substrates’
access
I
S1
S2
S & I bind to same site

57. Competitive inhibition

58. Non-competitive / Allosteric Inhibitors
“other” “site”
Distorts the conformation
of the enzyme
Negative
allosteric
regulator

59. Allosteric inhibition

60. Uncompetitive Inhibitors

61. Positive allosteric regulators
Helps enzyme work better
promotes/stabilizes an “active” conformation

62. Allosteric activation

63. Allosteric regulators change the shape
conformation of the enzyme
Stabilized inactive
form
Allosteric activater
stabilizes active from
Allosteric enyzme
with four subunits
Active site
(one of four)
Regulatory
site (one
of four)
Active form
Activator
Stabilized active form
Allosteric activater
stabilizes active form
Inhibitor
Inactive form
Non-
functional
active
site
(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors
dissociate when at low concentrations. The enzyme can then oscillate again.
Oscillation
Figure 8.20

64. A frequent regulatory modification
of enzymes
Phosphorylation

65. inactive
+ P
active
Phosphorylase kinase