Enzymes lecture.ppt

INTRODUCTION TO ENZYMES
1
DR HUSEINI WIISIBIE ALIDU
hwalidu@uhas.edu.gh
wiisibie@yahoo.com

Catalyst
• substance that increase rates of a
chemical reaction
• does not effect equilibrium
• remain unchanged in overall
process
• reactants bind to catalyst,
products are released
2

3
• Enzymes are biological catalysts.
• Recall that by definition, catalysts alter the
rates of chemical reactions but are neither
formed nor consumed during the reactions
they catalyze.
• Enzymes are the most sophisticated
catalysts known.
• Most enzymes are proteins. Some nucleic
acids exhibit enzymatic activities (e.g.,
rRNA). We will focus primarily on protein-
type catalysts.

Catalysts increase product formation by
(1) lowering the energy barrier (activation energy)
for the product to form
(2) increases the favorable orientation of
colliding reactant molecules for product
formation to be successful (stabilize transition
state intermediate)
4

5
Thermodynamics governs enzyme reactions, just
the same as with other chemical reactions.
Gibb’s “Free Energy,” ΔG, determines the
spontaneity of a reaction:
• ΔG must be negative for a reaction to occur
spontaneously (“exergonic”).
• A system is at equilibrium and no net change can
occur if ΔG is zero.
• A reaction will not occur spontaneously if ΔG is
positive (“endergonic”); to proceed, it must
receive an input of free energy from another
source.

For the reaction: A + B → C + D,
ΔG = ΔGo + RT ln [C][D]
[A][B]
ΔG = ΔGo + RT ln Keq
• At 25°C, when Keq changes by 10-fold, ΔG
changes by only 1.36!
• Small changes in ΔG describe HUGE
changes in Keq.
Note: ΔGo’ or ΔG’ denotes pH=7

7
Δ
G
Δ
G
ΔG‡ ΔG‡
Exergonic Reaction:
(Spontaneous)
Endergonic Reaction:
(Non-spontaneous)
ΔG determines SPONTANEITY (“-”
for spontaneous)
ΔG‡ determines the RATE of the
reaction.

8
Enzymes – Activation Energy
Uncatalyzed Reaction: Catalyzed Reaction:
Lower activation energy (ΔG‡) increases the rate of reaction,
reaching equilibrium faster.
In this case, ΔG remains unchanged. Thus, the final ratio of
products to reactants at equilibrium is the same in both cases.
ΔG
‡
ΔG
‡
Δ
G
Δ
G

9
Enzymes – Gibbs Free Energy

10
• In biochemistry, we use slightly
different terms for the participants in a
reaction:
Traditional Biochemistry
Reactant Substrate
Catalyst Enzyme
Product Product

Catalytic Power
• Enzymes can accelerate reactions
as much as 1016 over uncatalyzed
rates!
• Urease is a good example:
– Catalyzed rate: 3x104/sec
– Uncatalyzed rate: 3x10 -10/sec
– Ratio is 1x1014 !
11

Specificity
• Enzymes selectively recognize
proper substrates over other
molecules
• Enzymes produce products in very
high yields - often much greater
than 95%
• Specificity is controlled by
structure - the unique fit of
substrate with enzyme controls the
selectivity for substrate and the
product yield 12

Classes of enzymes
1. Oxidoreductases = catalyze oxidation-
reduction reactions (NADH)
2. Transferases = catalyze transfer of functional
groups from one molecule to another.
3. Hydrolases = catalyze hydrolytic cleavage
4. Lyases = catalyze removal of a group from or
addition of a group to a double bond, or other
cleavages involving electron rearrangement.
5. Isomerases = catalyze intramolecular
rearrangement.
6. Ligases = catalyze reactions in which two
molecules are joined.
Enzymes named for the substrates and type of
reaction
13

PROSTHETIC GROUPS
• Many enzymes contain small nonprotein
molecules and metal ions that participate
directly in substrate binding or catalysis.
Termed prosthetic groups, cofactors, and
coenzymes.
• Prosthetic groups are distinguished by their
tight, stable incorporation into a protein’s
structure by covalent or noncovalent forces e.g.
pyridoxal phosphate, flavin mononucleotide
(FMN), flavin dinucleotide (FAD), thiamin
pyrophosphate, biotin, and the metal ions of Co,
Cu, Mg, Mn, Se, and Zn (metalloenzymes).
15

COFACTORS
• They bind in a transient, dissociable manner
either to the enzyme or to a substrate such as
ATP.
• Cofactors must be present in the medium
surrounding the enzyme for catalysis to occur.
• The most common cofactors also are metal ions.
• Enzymes that require a metal ion cofactor are
termed METAL-ACTIVATED ENZYMES to
distinguish them from the METALLOENZYMES
for which metal ions serve as prosthetic groups.
16

COENZYMES
• They serve as recyclable shuttles—or group
transfer reagents—that transport many
substrates from their point of generation to
their point of utilization.
• Association with the coenzyme also stabilizes
substrates such as hydrogen atoms or hydride
ions.
• Other substance transported are methyl groups
(folates), acyl groups (coenzyme A), and
oligosaccharides (dolichol) – thiamin, riboflavin,
niacin, biotin
• Enzyme + Co-enzyme = holoenzyme
• Enzyme alone = apoenzyme 17

18
• For enzymes to function, they must come in
contact with the substrate.
• While in contact, they are referred to as
the “enzyme-substrate complex.”
• The high specificity of many enzymes led to
the hypothesis that enzymes were similar to
a lock… and the substrate was like a key:
(Fischer, 1890)
• In 1958, Koshland proposed that the enzyme
changes shape to fit the incoming substrate.
This is called an “induced fit.”

19
“Lock & Key” Theory:
“Induced Fit”
Theory:

20
• Enzymes are often quite large compared to
their substrates. The relatively small region
where the substrate binds and catalysis takes
place is called the “active site.” (e.g., human
carbonic anhydrase:)

21
• General Characteristics of Active Sites:
– The active site takes up a relatively small
part of the total volume of an enzyme
– The active site is a 3-dimensional
– cleft or crevice.
– Water is usually excluded unless it is a
reactant.
– Substrates bind to enzymes by multiple
weak attractions (electrostatic interactions,
hydrogen bonds, hydrophobic interactions,
etc.
– Specificity of binding depends on precise
spatial arrangement of atoms in space.

Kinetics
• study of reaction rate
• determines number of steps involved
• determines mechanism of reaction
• identifies “rate-limiting” step
22

23
• In 1913, two women scientists, Leonor
Michaelis and Maud Menten proposed a simple
model to account for the kinetic
characteristics of enzymes*.
Leonor
Michaelis?
Dr. Maud Menten

24
What was Michaelis’ and Menton’s contribution?
Since the enzyme and substrate must form the ES complex
before a reaction can take place, they proposed that the rate
of the reaction depended upon the concentration of ES:
E + S ES E + P
k1
k-1
k2
k-2
They also proposed that at the beginning of the reaction, very
little product returned to form ES. Therefore, k-2 was
extremely small and could be ignored:
E + S ES E + P
k1
k-1
k2

25
E + S ES E + P
k1
k-1
k2
k-2

26
E + S ES E + P
k1
k2
k3
The rate (Velocity) of the appearance of product, depends on [ES]:
V = k3[ES]
ES has two fates:
1. Go to product
2. Reverse back enzyme + substrate
When the catalyzed reaction is running smoothly and producing product
at a constant rate, the concentration of ES is constant at we say that
the reaction has reached a “steady state.” Therefore, we may say
that the rates for formation of ES and the breakdown of ES are
equal:
Rate of ES Formation d[ES]/dt = k1[E][S]
Rate of ES Breakdown -d[ES]/dt = k2[ES] + k3[ES]
At the “steady state:” d[ES]/dt = 0 = k1[E][S] – (k2+k3)[[ES]
Rearranging: k1[E][S] = (k2+k3)[[ES]

27
Steady State: k1[E][S] = (k2+k3)[[ES]
Rearrange, solving for [ES]: [ES] = [E][S] k 1 .
k2 + k3
Define M&M constant: Km: .. Km = k2 + k3 .
(“Dissociation”) k1
Result: [ES] = [E][S] / Km
If: [E] <<<[S], then [S] – [ES] ≈ [S]
Since: [Et] = [E] + [ES], it follows that [E] = [Et] – [ES]
Substituting for [E]: [ES] = ([Et] – [ES]) [S] / Km
Solving for [ES]: [ES] = [Et][S] / Km .
1+ [S] / Km
Simplifying: [Es] = [Et] [S]
[S] + Km

28
Steady State: k1[E][S] = (k2+k3)[[ES]
Rearrange, solving for [ES]: [ES] = [E][S] k 1 .
k2 + k3
Define M&M constant: Km:. Km = k2 + k3 .
k1
Result: [ES] = [E][S] / Km
If: [E] <<<[S], then [S] – [ES] ≈ [S]
Since: [Et] = [E] + [ES], it follows that [E] = [Et] – [ES]
Substituting for [E]: [ES] = ([Et] – [ES]) [S] / Km
Solving for [ES]:* [ES] = [Et][S] / Km .
1+ [S] / Km
Simplifying:* [Es] = [Et] [S]
[S] + Km
*Class Assignment: Show this algebreic rearrangement. Submit during next lecture period.

29
Now that we have an expression V = k3 [ES]
for [ES], we substitute into our V = k3 [Et] [S] .
“velocity” equation: [S] + Km
Consider [S] and Km: V = k3 [Et] [S] .
[S]+Km
As [S] → ∞, then [S] → 1
[S]+Km
We can define maximal velocity Vmax = k3 [Et]
as the velocity when [S] = ∞.
(We also assume that under these conditions, all enzymes [Et] are bound to S in the ES complex. )
The rate constant, k3, is the “turnover number,” or the maximum number of
substrates can be converted to products by a single enzyme molecule.
Therefore: V = Vmax [S]
(M&M Equation) [S] + Km

30
(M&M Equation) V = Vmax [S]
[S] + Km
What does this equation describe?
• It describes the velocity of an enzyme-catalyzed reaction at different
concentrations of substrate [S].
• It helps determine the maximum velocity of the catalyzed reaction.
• It assigns a value for Km, the “Michaelis constant,” that is inversely
proportional to the affinity of the enzyme for its substrate.
How is this equation utilized in the laboratory?
• A series of test tubes are prepared, all with identical concentrations of
enzyme, but increasing concentrations of substrate.
• The velocity of each tube increases as the substrate increases.
• A plot of the results is hyperboic, reaching an asymptote we define as
Vmax.

31
Why does the velocity reach a maximum?
V = Vmax [S]
[S] + Km

32
The Michaelis-Menton
equation was a pivotal
contribution to
understanding how
enzymes functioned.
However, during routine
use in the laboratory, it
was difficult to estimate
Vmax. Everyone had
different ideas the
actual value for Vmax.
Since it is impossible to
actually make a solution
with infinite
concentration of
substrate, a different
equation was needed.

33
A relatively simple solution was provided by Lineweaver and Burke, who simply suggested
that the M&M equation be inverted. This would yield a “double inverse plot” that is
linear:
(M&M Equation) V = Vmax [S]
[S] + Km
Inverting the Equation yields: 1 = Km 1 + 1 .
(Lineweaver-Burke Equation) V Vmax [S] Vmax
By plotting 1/ V as a function of 1/[S],
a linear plot is obtained:
Slope = Km/Vmax
y-intercept = 1/Vmax

34
Comparision of these two methods of plotting the same data:
Michaelis-Menton Equation: Linewaver-Burke Equation:

36
Factors Affecting Activity
Temperature affects enzyme activity. Higher
temperatures mean molecules are moving
faster and colliding more frequently.
Up to a certain point, increases in temperature
increase the rates of enzymatic reactions.
Excess heat can denature the enzyme, causing
a permanent loss of activity.
Examples:
• Cooking denatures many enzymes, killing
bacteria and inactivating viruses, parasites,
etc.
• Grass grows faster during the hot summer
than during the cooler spring or fall.
• Insects cannot move as fast in cold
weather as they can on a hot day.
• Operating rooms are often cooled down to
slow a patient’s metabolism during surgery.

37
pH often affects enzymatic reaction rates. The “optimum pH” refers to the pH
at which the enzyme exhibits maximum activity. This pH varies from enzyme
to enzyme:

Km = [S] @ ½ Vmax
(units moles/L=M)
(1/2 of enzyme bound to S)
Vmax = velocity where all of the
enzyme is bound to substrate
(enzyme is saturated with S)
38

What does Km mean?
1. Km = [S] at ½ Vmax
2. Km is a combination of rate constants
describing the formation and breakdown of
the ES complex
3. Km is usually a little higher than the
physiological [S]
39

Limitations of M-M
1. Some enzyme catalyzed rxns show more complex behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2. Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2
Must optimize one substrate then calculate kinetic
parameters for the other
3. Assumes k-2 = 0
4. Assume steady state conditions
40

Enzyme Inhibition
• Inhibitor – substance that binds to an enzyme and interferes
with its activity
• Can prevent formation of ES complex or prevent ES
breakdown to E + P.
• Irreversible and Reversible Inhibitors
• Irreversible inhibitor binds to enzyme through covalent
bonds (binds irreversibly)
• Reversible Inhibitors bind through non-covalent interactions
(disassociates from enzyme)
• Why important?
41

Enzyme Inhibitor Types
• Inhibitors of enzymes are generally
molecules which resemble or mimic a
particular enzymes substrate(s). Therefore,
it is not surprising that many therapeutic
drugs are some type of enzyme inhibitor.
The modes and types of inhibitors have been
classified by their kinetic activities and sites
of actions. These include Reversible
Competitive Inhibitors, Reversible Non-
Competitive Inhibitors, and Irreversible
Inhibitors

Reversible Inhibitors
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
• Competitive
• Uncompetitive
• Non-competitive
43

Types of Reversible Enzyme
Inhibitors
44

Competitive Inhibitor (CI)
•CI binds free enzyme
•Competes with substrate for enzyme binding.
•Raises Km without effecting Vmax
•Can relieve inhibition with more S
45

46
The antibiotic sulfanilamide was first discovered in 1932. Sulfanilamides and its
derivatives are called “sulfa drugs.”
Sulfanilamide is structurally similar to p-aminobenzoic acid (PABA), that is
required by many bacteria to produce an important enzyme cofactor, folic acid.
Sulfanilamide acts as a competitive inhibitor to enzymes that convert PAGA
into folic acid, resulting in a depletion of this cofactor. This results in
retarded growth and eventual death of the bacteria. (Mammals absorb their
folic acid from their diets, so sulfanilamide exerts no effects on them.)

47
By adding various functional groups to the basic structure,
increased effectiveness has been achieved:

48
Methotrexate is a competetive inhibitor for the coenzyme tetrahydrofolate
(required for proper activity of the enzyme dihydrofolate reductase). This
enzyme assists in the biosynthesis of purines and pyrimidines.
Methotrexate binds 1,000-fold more tightly to this enzyme than tetrahydrofolate,
significantly reducing nucleotide base synthesis. It is used to treat cancer.

Uncompetitive Inhibitor (UI)
•UI binds ES complex
•Prevents ES from proceeding to E + P or back to E + S.
•Lowers Km & Vmax, but ratio of Km/Vmax remains the same
•Occurs with multisubstrate enzymes
49

Non-competitive Inhibitor (NI)
•NI can bind free E or ES complex
•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t effect Km
•Alters conformation of enzyme to effect catalysis but not
substrate binding 50

• Irreversible inhibitors generally result in the destruction
or modification of an essential amino acid required for
enzyme activity.
•
• Frequently, this is due to some type of covalent link
between enzyme and inhibitor.
• These types of inhibitors range from fairly simple,
broadly reacting chemical modifying reagents (like
iodoacetamide that reacts with cysteines) to complex
inhibitors that interact specifically and irreversibly with
active site amino acids. (termed suicide inhibitors).
Irreversible Inhibitors

• These inhibitors are designed to mimic the
natural substrate in recognition and binding
to an enzyme active site.
• Upon binding and some catalytic
modification, a highly reactive inhibitor
product is formed that binds irreversibly and
inactivates the enzyme.
• Use of suicide inhibitors have proven to be
very clinically effective

53
Enzymes – Inhibition
Irreversible Inhibitors are toxic. In the laboratory they can be used to map the
active site. These inhibitors often form covalent linkages to amino acids at the
active site.
DIPF (diisopropylphosphofluoridate) forms a covalent linkage to serine. If serine
plays an important catalytic role for the enzyme, DIPF can permanantly disable
the enzyme. Acetycholinesterase is an excellent example of DIPF inactivation
(making agents such as DIPF potent nerve agents):

54
Enzymes – Inhibition
Another example of irreversible inhibition by covalent modification
is the reaction between iodoacetamide and a critical cysteine
residue:

55
Enzyme Inhibition – Penicillin
Penicillin is a classic irreversible enzyme inhibitor, acting on bacterial
“transpeptidase.” This enzyme strengthens bacterial cells walls, by
forming peptide bonds between D-amino acids that cross link the
peptidoglycan structure in cell walls.
Penicillin contains a beta-lactam ring (cyclic amide) fused to a thiazolidine
ring:

56
Penicillin’s structure is VERY SIMILAR to the normal
substrate for this enzyme.
In fact, penicillin is drawn into the active site of the
transpeptidase enzyme much like a competetive
inhibitor would be, due to its structural similarity:

57
Upon binding to the active site, the beta-lactam ring
opens and forms a covalent linkage to a serine at the
active site, permanently deactivating the enzyme:

Biochemistry 3070 – Enzymes 58
Over the years, organic
chemists altered the
basic penicillin molecule,
adding groups for
better acid resistance
and a broader
antibacterial activity
spectrum.
“PenVK” is the trade name
for
“Penicillin V, potassium
salt.”
Due to the structural
similarities between
these “cillins,” allergies
to one type of cillin,
extend throughout the
entire group of “beta-
lactams.”

Regulation of Enzyme Activity
Enzyme quantity – regulation of gene expression (Response time =
minutes to hours)
a) Transcription
b) Translation
c) Enzyme turnover
Enzyme activity (rapid response time = fraction of seconds)
a) Allosteric regulation
b) Covalent modification
c) Association-disassociation’
d) Proteolytic cleavage of proenzyme
60

Allosteric Regulation
• End products are often inhibitors
• Allosteric modulators bind to site other
than the active site
• Allosteric enzymes usually have 4o
structure
• Vo vs [S] plots give sigmoidal curve for
at least one substrate
• Can remove allosteric site without
effecting enzymatic action
61

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