Title: Introduction to enzymes
1Introduction 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.
4Energy diagram
transition state
activation energy
reactants
Energy
2 H2O2
products
?H
2 H2O O2
5To 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.
6Enzymatic reactions
enzymatic activation energy
Energy
2 H2O2
?H
2 H2O O2
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8Steps 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.
113. 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
124. Enzymes are not changed by the biochemical
reactions. Enzyme substrate ? ES ? enzyme
product They can be reused after the reaction.
135. 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.
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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)
17NAD (nicotinamide adenine dinucleotide)
reactive site
Nicotinamide (niacin)
adenine
ribose
18FAD (flavin adenine dinucleotide)
19Coenzyme 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
20Other 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
27Naming 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.
28Classification 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
29Example of each class of enzymes see Table 6.2.
30Kinetics 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.
31Effect 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.
33Effect of substrate concentration
Rate of reaction (velocity)
Substrate concentration
34Michaelis-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.
38Michaelis-Menten Equation vo Vmax .
S KM S vo initial velocity Vmax is
maximum velocity S substrate concentration
39When 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.
40k3
k1
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
42Theoretically, 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.
44Lineweaver-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
45Lineweaver-Burk equation
- An alternate approach to determine Vmax
46Lineweaver-Burk Curve Plot 1/ vo against
1/S, you get a linear line.
47Lineweaver-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
49Vmax 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
50When Vo ½ Vmax, S ?
When Vo ½ Vmax, S Km 0.001M When Vo
1/3 Vmax, S ?
51KM 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
53Characteristics 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.
54Binding 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.
55Lock and key model
- This model assumes that an enzyme active site
will only accept a specific substrate.
56Induced fit model
- This new model recognizes that there is much
flexibility in an enzymes structure. - An enzyme is able to conform to a substrate.
57Skip P158 p160 (Acid-base Catalysis, Metal ion
Catalysis, and Covalent Catalysis)
58Factors 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.
59c. 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
61Examples of optimum pH
- Enzyme Source pH
- pepsin gastric mucosa 1.5
- sucrase intestine 6.2
- catalase liver 7.3
- arginase beef liver 9.0
62Effect 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
63Other factors, cofactor, coenzyme and modulators.
64Enzyme 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.
65Reversible 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.
66Example 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).
67Neuromuscular 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..
69Reversible 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
70a. 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
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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
76c. 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.
78The 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.