2. What are Enzymes?
• Enzymes are catalytically active biological
macromolecules
• Specific, Efficient, Active in Aqueous Solution
• Most enzymes are globular proteins,
however some RNA (ribozymes, and ribosomal RNA) also
catalyze reactions
Non-peptide Co-Factors (Metals, Vitamins, Coenzymes)
• Enzymes can be classified functionally
4. Why Biocatalysis?
• Higher reaction rates
• Greater reaction specificity
• Milder reaction conditions
• Capacity for regulation
- -
COO COO
NH2
-
-
O COO
• Metabolites have
OH COO
many potential
-
pathways of
-
O COO
Chorismate
COO
- decomposition
COO OH mutase -
OOC
O
• Enzymes make the
desired one most
OH
NH2
favorable
5. Enzymatic Substrate Selectivity
OH
H
H
- +
OOC NH3 - +
OOC NH3
H
-
OOC
+
NH3
No binding
OH
HO OH
H
H Binding but no
H
NH
CH3
reaction
Example: Phenylalanine hydroxylase
6.
7. Enzyme Catalysis
• Enzyme: a biological catalyst
• with the exception of some RNAs that
catalyze their own splicing (Section 10.4), all
enzymes are proteins
• enzymes can increase the rate of a reaction
by a factor of up to 1020 over an uncatalyzed
reaction
• some enzymes are so specific that they
catalyze the reaction of only one
stereoisomer; others catalyze a family of
similar reactions
• The rate of a reaction depends on its
activation energy, ∆G°‡
• an enzyme provides an alternative pathway
with a lower activation energy
9. Temperature dependence of catalysis
• Temperature can also
catalyze reaction (increase
rate)
• This is dangerous, why?
• Increasing temperature will
eventually lead to protein
denaturation
11. Why Study Enzyme Kinetics?
• Quantitative description of biocatalysis
• Determine the order of binding of substrates
• Elucidate acid-base catalysis
• Understand catalytic mechanism
• Find effective inhibitors
• Understand regulation of activity
12.
13. Initial Rates, v0
• Linear region
• [S]≅[S]0
• [P] ≅ 0
• Enzyme kinetics
saturable
• V0 = Vmax when [S]=
∞
14. Michaelis-Menten Model
• For an enzyme-catalyzed reaction
k1 k2
E + S ES P
k-1
• The rates of formation and breakdown of ES are
given by these equations
rate of formation of ES = k1 [E][S]
rate of breakdown of ES = k-1 [ES] + k 2[ES]
• At the steady state
k1 [E][S] = k -1[ES] + k 2[ES]
15. Michaelis-Menten Model (Cont’d)
• When the steady state is reached, the concentration
of free enzyme is the total less that bound in ES
[E] = [E] T - [ES]
• Substituting for the concentration of free enzyme and
collecting all rate constants in one term gives
([E]T - [ES]) [S] k-1 + k2
= = KM
[ES] k1
• Where KM is called the Michaelis constant
16. Michaelis-Menten Model (Cont’d)
• It is now possible to solve for the concentration of the
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
= KM
[ES]
[E]T [S] - [ES][S] = KM[ES]
[E]T [S] = [ES](K M + [S])
• Or alternatively [E] T [S]
[ES] =
KM + [S]
17. Michaelis-Menten Model (Cont’d)
• In the initial stages, formation of product depends only on the
rate of breakdown of ES
k 2[E]T [S]
Vinit = k2 [ES] =
KM + [S]
• If substrate concentration is so large that the enzyme is
saturated with substrate [ES] = [E]T
Vinit = Vmax = k2 [E]T
• Substituting k2[E]T = Vmax into the top equation gives
Vmax [S] Michaelis-Menten
Vinit =
KM + [S] equation
19. Linearizing The Michaelis-Menten Equation
• It is difficult to determine Vmax experimentally
• The equation for a hyperbola
Vmax [S]
V= (an equation for a hyperbola)
KM + [S]
• Can be transformed into the equation for a straight line by taking
the reciprocal of each side
1 = KM + [S] KM [S]
= +
V Vmax [S] Vmax [S] Vmax [S]
1 = KM 1
+
V Vmax [S] Vmax
20. Lineweaver-Burk Plot
• The Lineweaver-Burke plot has the form y = mx + b, and is the
formula for a straight line
1 KM 1 1
= • +
V Vmax [S] Vmax
y = m • x + b
• a plot of 1/V versus 1/[S] will give a straight line with slope of
KM/Vmax and y intercept of 1/Vmax
• such a plot is known as a Lineweaver-Burk double reciprocal
plot
21. Lineweaver-Burk Plot (Cont’d)
• KM is the dissociation constant for ES; the greater the value of KM,
the less tightly S is bound to E
• Vmax is the turnover number
22. Turnover Numbers
• Vmax is related to the turnover number of enzyme:also
called kcat
Vax
=t r oe n me
m
un v r_ u br=kt
ca
E]
[ T
• Number of moles of substrate that react to form product
per mole of enzyme per unit of time
23.
24.
25. Chapter Seven
The Behavior of
Proteins:
Enzymes, Mechanisms,
and Control
26. Enzymes fall into classes based on function
• There are 6 major classes of enzymes:
1.Oxidoreductases which are involved in oxidation,
reduction, and electron or proton transfer reactions;
2.Transferases, catalysing reactions in which groups
are transferred;
3.Hydrolases which cleave various covalent bonds by
hydrolysis; 4
4.Lyases catalyse reactions forming or breaking
double bonds;
5.Isomerases catalyse isomerisation reactions;
6.Ligases join substituents together covalently.
27. Allosteric Enzymes
• Allosteric: Greek allo + steric, other shape
• Allosteric enzyme: an oligomer whose biological activity is affected by
other substances binding to it
• these substances change the enzyme’s activity by altering the
conformation(s) of its 4°structure
• Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an
• allosteric inhibitor
• allosteric activator
• Aspartate transcarbamoylase (ATCase)
• feedback inhibition
30. Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
• Irreversible inhibitors (inactivators) react with the enzyme
- one inhibitor molecule can permanently shut off one enzyme molecule
- they are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to, and can dissociate from the enzyme
- they are often structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme
• Reversible inhibitor can bind:
– To the free enzyme and prevent the binding of the
substrate
– To the enzyme-substrate complex and prevent the
reaction
32. Competitive Inhibition
Enzyme
S
I
In competitive inhibition,
the inhibitor competes
with the substrate for the
same binding site
33. Competitive inhibitors
• Enzymes can be inhibited competitively, when the
substrate and inhibitor compete for binding to the
same active site
• This can determined by plotting enzyme activity with
and without the inhibitor present.
• Competitive Inhibition
• In the presence of a competitive inhibitor, it takes a
higher substrate concentration to achieve the same
velocities that
• were reached in its absence. So while Vmax can still
be reached if sufficient substrate is available, one-
half Vmax requires a higher [S] than before and thus
Km is larger.
34. Competitive Inhibition
- Reaction Mechanism
E+S ES E+P
+
I
In competitive inhibition,
EI the inhibitor binds only to
the free enzyme, not to
the ES complex
35. General Michaelis-Menten Equation
Vmax,app [S]
v=
Km,app + [S]
This form of the Michaelis-Menten
equation can be used to understand how
each type of inhibitor affects the reaction
rate curve
36. In competitive inhibition, only the apparent Km is
affected (Km,app> Km),
The Vmax remains unchanged by the presence of
the inhibitor.
37. Competitive inhibitors alter the
.
apparent Km, not the Vmax
- Inhibitor
Vmax
Reaction Rate
+ Inhibitor
Vmax
2
Vmax,app = Vmax
Km,app > Km
Km Km,app
[Substrate]
38. The Lineweaver-Burk plot is diagnostic for
competitive inhibition
1 = Km,app 1
+ 1 Increasing [I]
v Vmax [S] Vmax
Km,app
1 Slope =
Vmax
v
1
Vmax
-1 1
Km,app
[S]
39. Relating the Michaelis-Menten equation, the v vs. [S] plot,
and the physical picture of competitive inhibition
Inhibitor
S
.
competes with
substrate, - Inhibitor
Vmax
.
decreasing its I
Reaction Rate
apparent affinity: + Inhibitor
Km,app > Km Vmax
2
Km,app > K m
E+S ES E+P V max,app = V max
+
I Km Km,app
Formation of EI
Formation of EI [Substrate]
complex shifts reaction
complex shifts reaction
to the left: K m,app > K
EI to the left: Km,app > Kmm
40. Noncompetitive Inhibition
.
I I
S
Enzyme S Enzyme
the inhibitor
does not
interfere with
S substrate
I I
binding (and
S vice versa)
Enzyme Enzyme
41. Non-competitive inhibitor
• With noncompetitive inhibition, enzyme molecules
that have been bound by the inhibitor are taken out of
the game so enzyme rate (velocity) is reduced for all
values of [S], including Vmax and one-half Vmax but
• Km remains unchanged because the active site of
those enzyme molecules that have not been inhibited
is unchanged.
42. Noncompetitive Inhibition - Reaction
Mechanism
E+S ES E+P
+ + In noncompetitive
inhibition, the
I I inhibitor binds
enzyme
irregardless of
whether the
EI + S ESI substrate is
bound
43. Noncompetitive inhibitors decrease
.
the Vmax,app, but don’t affect the Km
Vmax - Inhibitor
Reaction Rate
Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app
Vmax,app < Vmax
Km,app = Km
Km [Substrate]
Km,app
44. Why does Km,app = Km for
noncompetitive inhibition?
E+S ES E+P
+ + The inhibitor binds
equally well to free
I I enzyme and the ES
complex, so it doesn’t
alter apparent affinity
EI + S
of the enzyme for the
ESI substrate
45. The Lineweaver-Burk plot is diagnostic for
noncompetitive inhibition
1 = Km 1 1 Increasing [I]
+
v Vmax,app [S] Vmax,app
1 Slope =
Km
v Vmax,app
1
Vmax,app
-1 1
Km
[S]
46. Relating the Michaelis-Menten equation, the v vs. [S] plot,
and the physical picture of noncompetitive inhibition
I I
.
S
Enzyme S Enzyme
Inhibitor doesn’t interfere
with substrate binding,
Km,app = K m
S
I I
.
S Vmax - Inhibitor
Enzyme Enzyme
Reaction Rate
Vmax,app
E+S ES E+P 1 + Inhibitor
+ + Even at high
substrate 1
V
V
2 max
Km,app > Km
V max,app < V max
I I
Formation of EI
levels,
2 max,app
inhibitor still
Vmax,app = = Km
K m,app Vmax
complex shifts reaction
binds, Km Km,app
to the left: Km,app<>[ES]
Km
EI + S ESI [E] t [Substrate]
V max,app < V max
47. Irreversible Inhibition
In irreversible
Enzyme inhibition, the
inhibitor binds to
S the enzyme
O I irreversibly through
formation of a
covalent bond with
the enzyme ,
permanently
inactivating the
enzyme
48. Irreversible Inhibition - Reaction
Mechanism
E+S ES E+P
+ In irreversible
I inhibition, the inhibitor
permanently inactivates
the enzyme. The net
effect is to remove
EI enzyme from the
reaction.
Vmax decreases
No effect on Km
49. The Michaelis-Menten plot for an irreversible
inhibitor looks like noncompetitive inhibition
.
Vmax - Inhibitor
Reaction Rate
Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app
Vmax,app < Vmax
Km,app = Km
Km [Substrate]
Km,app
50. Irreversible inhibition is distinguished from
noncompetitive inhibition by plotting Vmax vs [E]t
.
tor
i bi
tor
Enzyme is
nh
tor
i
bi inactivated
hib
eI
Inhi
- In
until all of the
ibl
Vmax
ble itive
rsi et
rs
ve mp irreversible
ve
Re co
inhibitor is
rr e
+ n
No
+I used up
[E]t
[E]t < [I] [E]t > [I]
[E]t = [I]
51. Summary-Enzyme Inhibition
• Competitive Inhibitor
• Binds to substrate binding site
• Competes with substrate
• The affinity of the substrate appears to be decreased
when inhibitor is present (Km,app >Km)
• Noncompetitive inhibitor
• Binds to allosteric site
• Does not compete with the substrate for binding to the
enzyme
• The maximum velocity appears to be decreased in the
presence of the inhibitor (Vmax,app <Vmax)
• Irreversible Inhibitor
• Covalently modifies and permanently inactivates the
enzyme
55. Allosteric Enzymes
• Effector molecules change the activity of an
enzyme by binding at a second site
• Some effectors speed up enzyme action (positive
allosterism)
• Some effectors slow enzyme action (negative allosterism)
56. Protein Modification
• In protein modification a chemical group is
covalently added to or removed from the protein
• Covalent modification either activates or turns off the
enzyme
• The most common form of protein modification is
addition or removal of a phosphate group
• This group is located at the R group (with a free –
OH) of:
• Serine
• Threonine
• Tyrosine
57. Control of Enzyme Activity via Phosphorylation
• The side chain -OH groups
of Ser, Thr, and Tyr can
form phosphate esters
• Phosphorylation by ATP can
convert an inactive
precursor into an active
enzyme
• Membrane transport is a
common example
59. Proenzymes
• A proenzyme, an enzyme made in an inactive
form
• It is converted to its active form
• By proteolysis (hydrolysis of the enzyme)
• When needed at the active site in the cell
• Pepsinogen is synthesized and transported to the
stomach where it is converted to pepsin
60. Coenzymes
• Coenzyme: a nonprotein
substance that takes part in
an enzymatic reaction and is
regenerated for further
reaction
• metal ions- can behave as
coordination compounds.
(Zn2+, Fe2+)
• organic compounds, many
of which are vitamins or
are metabolically related to
vitamins (Table 7.1).
61. NAD+/NADH
• Nicotinamide adenine
dinucleotide (NAD+) is used
in many redox reactions in
biology.
• Contains:
1) nicotinamide ring
2) Adenine ring
3) 2 sugar-phosphate groups
62. NAD+/NADH (Cont’d)
• NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
• Nicotinamide ring is where reduction-oxidation
occurs
Editor's Notes
FIGURE 6-18d Structure of chymotrypsin. (PDB ID 7GCH) (d) A close-up of the active site with a substrate (mostly green) bound. Two of the active-site residues, Ser 195 and His 57 (both red), are partly visible. The hydroxyl of Ser 195 attacks the carbonyl group of the substrate (the oxygen is purple); the developing negative charge on the oxygen is stabilized by the oxyanion hole (amide nitrogens, including one from Ser 195 , in orange), as explained in Figure 6-21. In the substrate, the aromatic amino acid side chain and the amide nitrogen of the peptide bond to be cleaved (protruding toward the viewer and projecting the path of the rest of the substrate polypeptide chain) are in blue.
FIGURE 6-10 Initial velocities of enzyme-catalyzed reactions. A theoretical enzyme catalyzes the reaction S ↔ P, and is present at a concentration sufficient to catalyze the reaction at a maximum velocity, V max , of 1 μ M/min. The Michaelis constant, K m (explained in the text), is 0.5 μ M. Progress curves are shown for substrate concentrations below, at, and above the K m . The rate of an enzyme-catalyzed reaction declines as substrate is converted to product. A tangent to each curve taken at time = 0 defines the initial velocity, V 0 , of each reaction.
FIGURE 6-31 Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.