Transcript Classification of Enzymes

Chapter 5 Enzymes
What are enzymes?
and
How do they work?
5.1 Introduction to Enzymes
Enzymes are catalysts
What properties would an ideal catalyst have?
5.1 Introduction to Enzymes
What properties would ideal catalysts
have?
1. High degree of specificity for their substrates.
2. Accelerate chemical reactions tremendously.
3. Function in mild conditions.
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
A Few Definitions
Cofactor,
Coenzyme,
Prosthetic group
Holoenzyme
Apoenzyme
5.1 Introduction to Enzymes
Cofactor – additional chemical component needed for catalysis.
- often an inorganic metal ion (mineral).
5.1 Introduction to Enzymes
Coenzyme – complex organic molecule needed for catalysis.
- often a vitamin
5.1 Introduction to Enzymes
Prosthetic group – non amino acid portion of the enzyme
needed for catalysis. Often a coenzyme or metal ion.
Holoenzyme – complete catalytically active enzyme, with all
necessary prosthetic groups.
Apoenzyme – The protein part of the holoenzyme.
Prosthetic groups are absent.
5.1 Introduction to Enzymes
5.1 Introduction to Enzymes
Classification of Enzymes
5.1 Introduction to Enzymes
Classification of Enzymes
5.2 How Enzymes work
Consider the conversion of S  P (uncatalyzed reaction)
5.2 How Enzymes work
E + S  ES  EP  E + P
5.2 How Enzymes work
E + S  ES  EP  E + P
1. Enzymes affect the reaction rate,
not the equilibrium
2. Enzymes lower activation energy
3. Enzymes stabilize the transition state of a
reaction
5.2 How Enzymes work
Three mechanisms of catalysis
1. General Acid-Base Catalysis
2. Covalent catalysis
3. Metal Ion Catalysis
(1) General Acid-Base Catalysis
Charged intermediates formed during catalysis are stabilized by
donation or acceptance of protons by an amino acid side chain in
the active site
Amino acid side chains that can participate in acid-base catalysis
(2) Covalent catalysis
A transient covalent bond is formed between enzyme and
substrate during catalysis. After catalysis the covalent
complex is released and free enzyme is regenerated.
A-B + X:  A-X + B A + X: + B
Substrate
for catalysis
Amino Acid
side chain
Covalent
bond
Products
Amino Acid
side chain
(2) Metal Ion Catalysis
Metal ions bound to the enzyme participate in catalysis. The
metal may form an ionic interaction with a substrate or
mediate oxidation - reduction by donating or accepting
electrons.
5.3 Enzyme kinetic
Initial rates of reaction are measured in enzyme kinetics
E + S  ES  EP  E + P
The rate of reaction is dependent on substrate concentration
[S] – substrate concentration
Vo – initial velocity of a reaction. A significant amount of substrate
has not yet been converted to product.
Vmax – maximal velocity of a reaction. Addition of more substrate
will not increase the rate of the reaction.
Km – The concentration of substrate at which the rate of the reaction
is half-maximal
Michaelis-Menten equation
The double-reciprocal plot
Experimental determination of Vmax and Km
k1
k2
k-1
k-2
k3
E + S  ES  EP  E + P
•k represents a rate constant.
•The conversion of S to P is described by a series of rate
constants.
•The conversion of EP to E + P is often the rate-limiting
step of the reaction.
•k3 =kcat, turnover number.
•kcat has the units of sec-1 (molecules / second)
Turnover numbers vary widely
Catalytic efficiencies of enzymes.
kcat / Km = catalytic efficiency.
The kcat / Km value is limited by the rate E and S can
diffuse together in aqueous solution.
The diffusion-controlled limit is 1 x 109 M-1sec-1
5.4 Enzyme Substrate Interaction
5.4 Enzyme substrate interaction
Catalytic site
Where the reaction actually occurs.
Binding Site
 Area that holds substrate in proper place.
Enzymes uses weak, non-covalent interactions to hold the
substrate in place based on R group of amino acids.
Shape is complementary to the substrate and determines the
specificity of enzyme.
Sites are pockets or clefts on the enzyme surface
5.4 Enzyme substrate interaction
5.4 Enzyme substrate interaction
5.4 Enzyme substrate interaction
5.4 Enzyme substrate interaction
5.5 Factors Affecting Enzyme Activity
(1)
5.5 Factors Affecting Enzyme Activity
5.5 Factors Affecting Enzyme Activity
5.5 Factors Affecting Enzyme Activity
5.5 Factors Affecting Enzyme Activity
(2)
5.5 Factors Affecting Enzyme Activity
5.5 Factors Affecting Enzyme Activity
5.5 Factors Affecting Enzyme Activity
(3) Enzyme Inhibition
Enzyme activity is decreased by inhibitors.This is the
basis of many pharmaceutical agents.
Many substances can inhibit enzyme activity.
Inhibitors include substrate analogs, toxins, drugs,
metal complexes.
5.5 Factors Affecting Enzyme Activity
Two broad classes of inhibitors:
Irreversible and Reversible.
Irreversible: Forms covlent or very strong noncovalent bonds.
The sites of attack is an amino acid group that participates in
normal enzymatic reaction.
Reversible: Forms weak, noncovalent bonds that readily
dissociate from an enzyme. The enzyme is only inactive when the
inhibitor is present.
5.5 Factors Affecting Enzyme Activity
Reversible Enzyme Inhibition
Competitive - the inhibitor competes with substrate
for binding to the active site of the enzyme
Uncompetitive – binds at a site on the enzyme
distinct from the active site–binds to ES complex
Mixed - binds to E or ES complex
5.5 Factors Affecting Enzyme Activity
(a) Competitive Inhibition
As [I] increases
Km increases
Vmax is unaffected
5.5 Factors Affecting Enzyme Activity
(b) Uncompetitive Inhibition
As [I] increases
Vmax decreases
Km decreases
5.5 Factors Affecting Enzyme Activity
(c ) Mixed Inhibition
As [I] increases
Vmax decreases
Km increases
Irreversible Enzyme Inhibition
An inhibitor forms an irreversibly covlent or very strong noncovalent
with the enzyme or destroys a functional group essential for catalytic
activity
Diisopropylfluorophosphate (DIFP) irreversibly
inhibits chymotrypsin
Exercises
•
1. One of the enzymes involved in glycolysis,
aldolase, requires Zn2+ for catalysis. Under
conditions of zinc deficiency, when the enzyme may
lack zinc, it would be referred to as the:
a)
b)
c)
d)
e)
substrate.
coenzyme.
holoenzyme.
prosthetic group.
apoenzyme.
Exercises
•
a)
b)
c)
d)
e)
2. Which one of the following is not among the
six internationally accepted classes of
enzymes?
Hydrolases
Ligases
Oxidoreductases
Polymerases
Transferases
5.1 Introduction to Enzymes
Classification of Enzymes
Exercises
•
3. Enzymes are potent catalysts because they:
a) are consumed in the reactions they catalyze.
b) are very specific and can prevent the conversion of
products back to substrates.
c) drive reactions to completion while other catalysts
drive reactions to equilibrium.
d) increase the equilibrium constants for the reactions
they catalyze.
e) lower the activation energy for the reactions they
catalyze
Exercises
•
a)
b)
c)
d)
e)
4. The role of an enzyme in an enzyme-catalyzed
reaction is to:
bind a transition state intermediate, such that it cannot
be converted back to substrate.
ensure that all of the substrate is converted to product.
ensure that the product is more stable than the
substrate.
increase the rate at which substrate is converted into
product.
make the free-energy change for the reaction more
favorable.
Exercises
•
a)
b)
c)
d)
e)
5. Which one of the following statements is true of
enzyme catalysts?
Their catalytic activity is independent of pH.
They are generally equally active on D and L isomers
of a given substrate.
They can increase the equilibrium constant for a given
reaction by a thousand fold or more.
They can increase the reaction rate for a given
reaction by a thousand fold or more.
To be effective, they must be present at the same
concentration as their substrate.
Exercises
•
a)
b)
c)
d)
e)
6. Which one of the following statements is true of
enzyme catalysts?
They bind to substrates, but are never covalently
attached to substrate or product.
They increase the equilibrium constant for a reaction,
thus favoring product formation.
They increase the stability of the product of a desired
reaction by allowing ionizations, resonance, and
isomerizations not normally available to substrates.
They lower the activation energy for the conversion
of substrate to product.
To be effective they must be present at the same
concentration as their substrates.
Exercises
• 7. Which of the following statements is false?
a) A reaction may not occur at a detectable rate
even though it has a favorable equilibrium.
b) After a reaction, the enzyme involved becomes
available to catalyze the reaction again.
c) For S P, a catalyst shifts the reaction
equilibrium to the right.
d) Lowering the temperature of a reaction will
lower the reaction rate.
e) Substrate binds to an enzyme's active site.
Exercises
•
a)
b)
c)
d)
e)
8. Enzymes differ from other catalysts in that
only enzymes:
are not consumed in the reaction.
display specificity toward a single reactant.
fail to influence the equilibrium point of the
reaction.
form an activated complex with the reactants.
lower the activation energy of the reaction
catalyzed.
Exercises
• 9. Which of the following statements about a plot of
V0 vs. [S] for an enzyme that follows MichaelisMenten kinetics is false?
a) As [S] increases, the initial velocity of reaction V0
also increases.
b) At very high [S], the velocity curve becomes a
horizontal line that intersects the y-axis at Km.
c) Km is the [S] at which V0 = 1/2 Vmax.
d) The y-axis is a rate term with units of mm/min.
Exercises
•
•
•
•
•
a)
b)
c)
d)
e)
10. Michaelis and Menten assumed that the overall reaction for
an enzyme-catalyzed reaction could be written as
k1
k2
E+S
ES
E+P
k-1
Using this reaction, the rate of breakdown of the enzymesubstrate complex can be described by the expression:
k1 ([Et] - [ES]).
k1 ([Et] - [ES])[S].
k2 [ES].
k-1 [ES] + k2 [ES].
k-1 [ES].
Exercises
•
11. Which of these statements about enzyme-catalyzed
a)
b)
c)
d)
reactions is false?
At saturating levels of substrate, the rate of an enzymecatalyzed reaction is proportional to the enzyme
concentration.
If enough substrate is added, the normal Vmax of a
reaction can be attained even in the presence of a
competitive inhibitor.
The rate of a reaction decreases steadily with time as
substrate is depleted.
The activation energy for the catalyzed reaction is the
same as for the uncatalyzed reaction, but the equilibrium
constant is more favorable in the enzyme-catalyzed
reaction.
Exercises
•
a)
b)
c)
d)
e)
12. In competitive inhibition, an inhibitor:
binds at several different sites on an enzyme.
binds covalently to the enzyme.
binds only to the ES complex.
binds reversibly at the active site.
lowers the characteristic Vmax of the enzyme.
Exercises
•
a)
b)
c)
d)
e)
13. Vmax for an enzyme-catalyzed reaction:
generally increases when pH increases.
increases in the presence of a competitive inhibitor.
is limited only by the amount of substrate supplied.
is twice the rate observed when the concentration of
substrate is equal to the Km.
is unchanged in the presence of a uncompetitive
inhibitor.
Exercises
14. Enzyme X exhibits maximum activity at pH = 6.9. X
shows a fairly sharp decrease in its activity when the pH
goes much lower than 6.4. One likely interpretation of
this pH activity is that:
•
a Glu residue on the enzyme is involved in the reaction.
•
a His residue on the enzyme is involved in the reaction.
•
the enzyme has a metallic cofactor.
•
the enzyme is found in gastric secretions.
•
the reaction relies on specific acid-base catalysis.
Exercises
•
.
15. Examples of cofactors include
A) Zn+2, Mg+2, and Ni+2.
B) biotin and thiamine pyrophosphate.
C) pyridoxal phosphate and coenzyme A.
D) b and c
E) a, b, and c
Exercises
•
16. Which are types of enzyme inhibition?
A) irreversible B) reversible C) temporary D) a, b, and c
E) a and b
Exercises
•
17. What type(s) of inhibition can be reversed?
A) competitive
B) non-competitive
C) mixed
D) all of the above
E) none of the above
Exercises
18. In this type of inhibition the enzyme can form a complex
with either the substrate (ES) or the inhibitor (EI), but
not both.
A) Competitive
B) Non-competitive
C) Mixed
D) All of the above
E) None of the above
Exercises
•
•
•
•
•
•
19. The site on the enzyme where the reaction occurs is
known as
a) Circe
b) active site
c) prosthetic group
d) substrates
Chymotrypsin cleaves
peptide bonds after
aromatic amino acid
side chains
Active site
amino acids
Aromatic amino acid
side chain binding pocket
Active site
of
chymotrypsin
Enzyme Mechanism
Steps in the cleavage
of a peptide bond
by chymotrypsin
The enzyme aspartate transcarbamoylase has six catalytic
subunits and six regulatory subunits
Catalytic
subunits
Regulatory
subunits