source = some mine, some Campbell 6th ed, some taken from public domain around the web.

1. Chapter Six
The Behavior of Proteins: Enzymes
Jody Haddow - UAEU

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

3. Carbonic Anhydrase
Tissues

 Lungs and Kidney
107 rate enhancement

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

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

8. Enzyme Catalysis (Cont’d)
• Consider the reaction
H2 O 2 H2 O + O2

9. Temperature dependence of catalysis
• Temperature can also
catalyze reaction (increase
rate)
• This is dangerous, why?
• Increasing temperature will
eventually lead to protein
denaturation

10. Michaelis-Menten Kinetics
• Initial rate of an enzyme-catalyzed reaction versus
substrate concentration

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

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

18. Michaelis-Menten Model (Cont’d)
• When [S]= KM, the equation reduces to
Vmax [S] Vmax [S] Vmax
V= = =
KM + [S] [S] + [S] 2

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

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

28. Feedback Inhibition
Formation of product
inhibits its continued
production

29. Allosteric Regulation; ATCase

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

31. Types of Inhibition
• Competitive Inhibition
• Noncompetitive Inhibition
• Irreversible Inhibition

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

52. Competitive/noncompetitive inhibitor

53. Effect of inhibitors

54. Enzyme Regulation
• Allosteric regulation,
• heterotropic ligand binding modulates
substrate binding and catalysis,
• Feedback regulates metabolic pathways
• Covalent modification – Reversible
• Phosphorylation, nucleotides, lipid anchors
• Proteolysis converts inactive pro-enzymes
(zymogens) to active

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

58. Covalent Modification
Lipase:

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