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Introduction to enzymes

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Title: Introduction to enzymes


1
Introduction to enzymes
  • Enzyme biological catalyst.
  • Permit reactions to go at conditions that the
    body can tolerate.
  • Typically are very large proteins.
  • Can process millions of molecules every second.
  • Very specific only react with one or a few
    types of molecules.

2
  • How does an enzyme work?
  • It accelerate a reaction by lowering activation
    energy.
  • All chemical reactions require a minimum
    energy input (activation energy) to get
    initiated.
  • The function of an enzyme is to lower the
    activation energy and speed up the reaction.

3
  • Consider the following reaction
  • 2 H2O2 2 H2 O O2
  • The reaction is thermodynamically favored but
    occurs very slowly.
  • Slow reaction rate is due to the high activation
    energy for the reaction.
  • Only a small portion of the molecules have
    sufficient energy to overcome this energy.

4
Energy diagram
transition state
activation energy
reactants
Energy
2 H2O2
products
?H
2 H2O O2
5
To increase the rate of a reaction, two
possibilities To increase the average energy
level of the reactant (H2O2) by increasing the
temperature, or to add a catalyst
(enzyme). Increasing temperature causes
increased molecule collision Not realistic for
living cells.
6
Enzymatic reactions
enzymatic activation energy
Energy
2 H2O2
?H
2 H2O O2
7
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8
Steps in an enzymatic reaction
  • 1. Enzyme and substrate combine to form a
    complex.
  • Complex goes through a transition state
  • not quite substrate or product
  • 3. A complex of the enzyme and the product is
    produced
  • 4. Finally the enzyme and product separate

9
  • Properties of Enzymes
  • Enzymes speed up reaction by lowering
  • activation energy
  • It forms a transient complex with reactant, thus
  • stabilize the transient state.
  • An enzyme does not change the position of the
  • reaction equilibrium, but increases the rate for
  • the equilibrium to be reached.
  • (The amount of the products is not increased)

10
 2. Enzymes are highly specific Each enzyme
usually catalyzes a single reaction.   Before
reaction substrate has to bind to the enzyme at
a specific site active site.   Enzyme
substrate ? ES ? enzyme product The active
site has to match with the substrate in shape
lock and key relationship.
11
3. Enzymes can be saturated Usually each enzyme
has only one or a few active sites, one on each
subunit. The concentration of substrate
molecules is usually higher than that of enzyme
active sites. Therefore, enzymes can be
saturated. A reaction can only reach certain
rate (Vmax) due to the limited enzyme active
sites
12
4. Enzymes are not changed by the biochemical
reactions. Enzyme substrate ? ES ? enzyme
product They can be reused after the reaction.

13
5. Enzymes may need coenzymes and cofactors
in order to function (p178 - p182)   Some
enzymes stay in an inactive form until binding
with a coenzyme or a cofactor   Cofactors are
ions such as Ca, Zn, Mg, Cu Coenzymes are
usually small organic molecules associated with
vitamins.   Apoenzyme Coenzyme ?
Holoenzyme (Inactive form) (or Cofactor)
(active form)  
14
Coenzyme Vitamin NAD Niacin FAD
Riboflavin Biotin Biotin Coenzyme
A Pentothenic acid Other vitamins related
with cozymes vitamin C, vitamin B family,
Table 7.1 on p177.
15
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16
  • Some enzymes require a second species to be
    present in order to do their job.
  • Cofactor - prosthetic group needed to
    activate the apoenzyme.
  • - usually a metal ion that holds
    protein in the proper shape.
  • Coenzymes are usually small organic molecules
  • associated with vitamins.
  •  
  • Apoenzyme Coenzyme ? Holoenzyme
  • (Inactive form) (or Cofactor) (active
    form)

17
NAD (nicotinamide adenine dinucleotide)
reactive site
Nicotinamide (niacin)
adenine
ribose
18
FAD (flavin adenine dinucleotide)
19
Coenzyme A
ADP
pantothenate unit
NH
2
O
H
CH3
N
N
O
P
O
P
C-CH2-CH2-N-C-C-C-CH2
O
N
-
-
N
CH
O
O
CH3
HO
H
H-N
2
O
CH2-CH2
SH
OH
O
-
P
O
O
-
O
20
Other water-soluble vitamins
  • Thiamine - Vitamin B1
  • Uses
  • Coenzyme thiamine pyrophosphate. Required for
    decarboxylation reactions in carbohydrate
    metabolism.

21
  • Pyridoxine - Vitamin B6
  • Found in fish, meat, green leafy vegetables.
  • Uses.
  • Coenzyme pyridoxal phosphate. Required in
    synthesis and breakdown of amino acids.

22
  • Folic acid a B vitamin.
  • Found in meat, cereals, green vegetables,
    intestinal bacteria.
  • Uses
  • Coenzyme tetrahydrofolate, needed for protein
    synthesis and the synthesis of purines and
    pyrimidines.

23
  • Biotin - B vitamin.
  • Found in liver, egg yolks, cheese, peanuts.
    Synthesized by intestinal bacteria.
  • Uses
  • Involved in carboxylation
  • and decarboxylation in
  • fats, carbohydrates, and proteins.

24
  • Vitamin B12
  • Found in meat, eggs, dairy products.
  • Uses
  • Coenzyme cobamide Important for the production
    of red
  • blood cells.
  • (Pernicious anemia)

25
  • Vitamin C - ascorbic acid
  • Found in fresh fruit and vegetables.
  • Uses
  • Formation and maintenance of collagen. Enhances
    absorption of iron from foods.
  • Serves as an antioxidant.

26
  • Lipid Soluble Vitamins
  • Vitamin A
  • Vitamin K
  • Vitamin D
  • Vitamin E

27
Naming of enzymes
  • Name is based on - what it reacts with
  • - how it reacts
  • - end with -ase
  • Examples
  • lactase - enzyme that reacts with lactose.
  • pyruvate decarboxylase - removes carboxyl
    from pyruvate.
  • Common name reactant ase.

28
Classification of enzymes
  • Based on type of reaction (table 6.1)
  • Oxidoreductase catalyze a reduction-oxidation
    reaction
  • Transferase transfer a functional group
  • Hydrolase cause hydrolysis reactions
  • Lyase cause formation of double bonds
  • Isomerases rearrange functional groups
  • Ligase join two molecules by forming
  • C-C, C-O, C-S, C-N
    bonds

29
Example of each class of enzymes see Table 6.2.
30
Kinetics of Enzymatic Reactions
  Michaelis-Menten Model (p147 p152) For
many enzymes, the rate of reaction, V, varies
with the substrate concentration S. When other
conditions are controlled, the higher the
substrate concentration, the higher the reaction
rate, but only to a certain point (Vmax). This
catalytic behavior is observed for most enzymes.

31
Effect of substrate concentration
Rate of reaction (velocity)
Substrate concentration
32
  • To plot a Michaelis-Menten curve
  • Increasing amount of substrate is added to a set
    of tubes containing equal amount of enzyme
  • The reaction rate is determined by measuring the
    quantity of the products in each tube.
  • A velocity curve is obtained by plotting the
    product formation against the quantity of
    substrate.

33
Effect of substrate concentration
Rate of reaction (velocity)
Substrate concentration
34
Michaelis-Menten Curve
When S is small, V is almost linearly
proportional to S. At high S, V is nearly
independent of S.
35
Michaelis-Menten Equation A mathematical model
that describes the relationship between the
reaction rate and substrate concentration. In
the simplest case, it involves the reaction of a
substrate (S) with an enzyme (E) to form an
activated complex (ES). The complex can then
decompose to a product (P) and the enzyme or
back to the substrate.  
36
  • E S ES P E
  • k1 the speed for the formation of ES
  • k2 the speed for the decomposition of ES
  • k3 the speed for the formation of product
  • k4 can be neglected because its effect is very
    small during the initial stages of the reaction.

37
  • Michaelis constant Km
  • KM also equals to S, when v 1/2 Vmax.
  • Unit of Km is molar or mM, same as the unit of
    S.

38
Michaelis-Menten Equation   vo Vmax .
S KM S   vo initial velocity Vmax is
maximum velocity S substrate concentration
39
When Km equals to S, the equation can be
rearranged to   vo Vmax S S
S Vmax S 2S  
Vmax 2   So, when Km equals to S, initial
velocity, Vo 1/2 Vmax.  
40
k3
k1
  • E S ES P E

k2
k4
41
Km k2 k3 k1 If k2 gtgt k3, the
equation can be written as Km k2
k1   Since E S E S, Km actually
describes the affinity between E and S. Km
- the dissociation constant Large KM weak ES
complex Small KM - Strong ES complex.  
k2
k1
42
Theoretically, Vmax and Km can be estimated from
a Michaelis-Menten curve, but is can be
difficult.
43
  • Using the Michaelis-Menten equation can be
    difficult to determine Vmax from experimental
    data.
  • An alternate approach was proposed by Lineweaver
    and Burk that results in a linear plot of data.

44
Lineweaver-Burk Equation Lineweaver and Burk
(1934) modified Michaelis -Menten's equation into
a more applicable way. If vo Vmax . S
Km S   1/ vo Km S Km
S Vmax . S Vmax
S Vmax . S Km . 1 1
Vmax S Vmax  
45
Lineweaver-Burk equation
  • An alternate approach to determine Vmax

46
Lineweaver-Burk Curve Plot 1/ vo against
1/S, you get a linear line.
47
Lineweaver-Burk equation
The intersection of the curve and the Y axis
represents 1/Vmax. The intersection point
on the X axis represents - 1/ Km. The slop
of the curve is Km / Vmax.
1/vo
1 / Vmax
slope of line KM / Vmax
-1 / KM
1 / S
48
  • Solve problem
  • 6.19 Assume that an enzyme has the following
  • kinetic constants
  • Vmax 50 umol/min
  • Km 0.001 M
  • Plot a Lineweaver-Burk curve to describe the
    reaction.
  • 2. Calculate the substrate concentration that
    will
  • yield a reaction rate of
  • a. ½ Vmax
  • b. 1/3 Vmax

49
Vmax 50, 1/Vmax 1/50 0.02 Km 0.001, 1/Km
1000
1/v
0.10 0.08 0.06 0.04 0.02
1/Vmax
1/Km
-1000 -800 -600 -400 -200 0 200 400
600 1000 1/S
50
When Vo ½ Vmax, S ?
When Vo ½ Vmax, S Km 0.001M When Vo
1/3 Vmax, S ?
51
KM Vmax
.


Km 0.001M, Vmax 50 umol/min, 1/Vo 1/3
Vmax 1 0.001 x 1 1
1/3 50 50 S 50 3
0.001 1 50 50S
50 2 0.001 50
50S 2S 0.001,
S 0.001/2 0.0005M
.



52
  • Today
  • Characteristics of Enzyme Binding Site
  • Factors That Affect Enzyme Activities
  • Enzyme Inhibition

53
Characteristics of Enzyme Active Sites
  • Catalytic site
  • Where the reaction actually occurs.
  • Binding site
  • Enzymes use weak, non-covalent interactions to
    hold the substrate in place.
  • Shape as a pocket or cleft, so that the
    substrates can fit in.

54
Binding between Enzyme and Substrate
  • Lock and key model
  • 1890 picture by Emil Fisher. This model assumed
    that only a substrate of the proper shape could
    fit with the enzyme.
  • Induced-fit model
  • Proposed by Daniel Koshland in 1958. This model
    assumes continuous changes in active site
    structure as a substrate binds.

55
Lock and key model
  • This model assumes that an enzyme active site
    will only accept a specific substrate.

56
Induced fit model
  • This new model recognizes that there is much
    flexibility in an enzymes structure.
  • An enzyme is able to conform to a substrate.

57
Skip P158 p160 (Acid-base Catalysis, Metal ion
Catalysis, and Covalent Catalysis)
58
Factors that affect enzyme activities a.
Substrate concentration   b. Enzyme
concentration     
The higher E, the higher the reaction rate,
as long as there are enough substrate.
59
c. pH optimal pH for most enzymes range from
6-8. Wrong pH ? protein denature.
Although there are some exceptions. d.
Temperature optimal temperature for
most enzymes range from 25 40oC. High
temperature ? denature
60
  • Effect of pH on Enzyme Activity

pepsin
vo
trypsin
2 4 6
8 10
pH
61
Examples of optimum pH
  • Enzyme Source pH
  • pepsin gastric mucosa 1.5
  • sucrase intestine 6.2
  • catalase liver 7.3
  • arginase beef liver 9.0

62
Effect of temperature on enzymatic reactions
Optimum temperature is usually 25 - 40oC but not
always.
temperature
  • Temperature exceeding normal range always reduces
    enzyme reaction rates.
  • T gt 53 C, most enzymes are denatured, but

63
Other factors, cofactor, coenzyme and modulators.
64
Enzyme inhibition (p160-_)
  • Many substances can inhibit enzyme activity.
  • Toxins, drugs, metal complexes can inhibit
    enzymes.
  • Inhibition studies can provide
  • Information on metabolic pathways.
  • Better understanding of enzyme reaction
    mechanisms.

65
Reversible and irreversible inhibitors
  • Irreversible
  • Inhibitors form very strong chemical bonds
    (covalent bonds) with the enzyme and dissociate
    from the target very slowly.
  • Reversible
  • Inhibitors form weak, noncovalent bonds that
  • readily dissociate from an enzyme. The
  • enzyme is only inactive when the inhibitor
  • is present.

66
Example of irreversible inhibition
  • Acetylcholinesterase and nerve gases
  • Acetylcholine is a neurotransmitter to cause
    muscle
  • contraction
  • Acetylcholinesterase destroys the acetylcholine
  • shortly after its been released to stop the
    signal.
  • The muscle is then relaxed
  • Nerve gases block the enzyme permanently.
  • Consistant neural stimulation can cause muscle
    spasm
  • (spastic paralysis).

67
Neuromuscular Junction
Acetylcholine binds with receptor causing
muscle contraction.
synaptic cleft
Ach
Ach-R
acetylcholinesterase - destroys excess
acetylcholine
68
  • Drugs and poisons that can inhibit
    Acetylcholinesterase
  • Diisopropyl fluorophosphates
  • Reacts with the serine residue at the active site
    of ACE, which can cause death..

69
Reversible Inhibition Inhibitors bind with
enzymes in a noncovalent Manner more often seen
in biosystems as part of normal metabolic
control.  Three types of reversible
inhibition Competitive inhibition Noncompetiti
ve inhibition Uncompetitive inhibition
70
a.     Competitive inhibition The inhibitor
resembles the structure of normal substrate and
competes for the binding site of the enzyme.
The inhibition depends on the concentration ratio
of substrate to inhibitor and the binding
affinity. A very high concentration of
substrate can overcome the effect of the
inhibitor, unless the inhibitor has much higher
affinity for the enzyme.  
71
  • Competitive Inhibitor
  • Resembles the normal substrate and competes with
    it for the same site.

normal substrate
competitive inhibitor
72
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73
  • Malonate, oxalate and pyrophosphate are
  • analogs of succunate. They can bind to
  • succinate dehydrogenase and competitively
    inhibit it.

74
  • b.     Noncompetitive inhibition
  • Both inhibitor and substrate bind simultaneously
  • to the enzyme on different sites.
  • The inhibitor does not prevent the binding of
  • the substrate.
  • It interferes with the catalytic function of the
  • enzymes.
  •  

75
  • Noncompetitive Inhibitors bind at a location
    other than the normal site.

noncompetitive site
inhibitor
76
c. Uncompetitive inhibition Similar to a
noncompetitive inhibition. The difference is
that an uncompetitive inhibitor only binds to
the ES complex, and slows down the
reaction.      
77
  • Uncompetitive inhibitors only bind to the ES
    complex.

78
The three types of reversible inhibition can be
differentiated by Lineweaver-Burk curves.
79
  • Competitive inhibitors decrease reaction rate
  • by increasing Km of the enzyme. Vmax not
  • changed
  • Noncompetitive inhibitors decrease reaction rate
  • by decrease Vmax. Km not changed.
  • Uncompetitive inhibitors decrease reaction rate
  • by changing both Km and Vmax, the slop of
  • the curve is not changed.

80
In real organisms, the catalytic activities of
many enzymes are regulated to meet different
physiological requirement. And the regulation
is accomplished mostly by competitive
inhibition.    
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