1) Enzymes require cofactors like vitamins and metal ions to function properly and carry out reactions. Common coenzymes include NAD+, FAD, coenzyme A, biotin, and vitamin B12. 2) NAD+ acts as an electron acceptor in many oxidation reactions, FAD carries electrons and hydrogen atoms, and coenzyme A transfers acyl groups. 3) The cofactors help enzymes by participating directly in chemical transformations and bringing reactive groups close together to reduce the activation energy of reactions. Living systems rely on cofactors to drive essential metabolic pathways.

1. ENZYME KINETICS

2. Enzymes
• Living organisms must be able to carry
out chemical reactions which are
thermodynamically very unfavorable
– Break and form covalent bonds
– Move large structures
– Effect three dimensional structure
– Regulate gene expression
• Do so through catalysis

3. 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 !

4. Effect of enzymes
• A common biological reaction can take
place without enzyme catalysis
– Will take 750,000,000 years
• With enzyme 22 milliseconds
• Even improvement of a factor of 1,000
would not be good enough
– Only 750,000 years
– Living systems would be impossible

5. 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

6. Substrate Specificity of Enzymes
• Almost all enzymes are globular proteins with one or more active sites on their
surface.
• The substrate is the reactant an enzyme acts on
• Reactants bind to the active site to form an enzyme-substrate complex.
• The 3-D shape of the active site and the substrates must match, like a lock and
key
• Binding of the substrates causes the enzyme to adjust its shape slightly, leading
to a better induced fit.
• When this happens, the substrates are brought close together and existing
bonds are stressed. This reduces the amount of energy needed to reach the
transition state.
Substate
Active site
Enzyme
Enzyme- substrate
complex

7. Stickase
From Lehninger; third edition

8. Enzymes
• Have immense importance in a number of
fields.
– Genetic diseases are frequently defects in
enzymes or increased/decreased levels of
enzymes
• Important diagnostic tools
– Drugs exert effects by interacting with enzymes
• MAO inhibitors
– Used in food processing and in chemical industry
– Enzyme inhibitors are a foundation of biological
weapons
– Genetic Engineering and Biotechnology use a lot
of enzymes!

9. Kinetics
• Is the science that describes the properties of
a chemical reaction including those mediated
by enzymes (catalysis)
• Measures the concentration of substrate
and/or products of a reaction to determine the
velocity of the reaction
• Measures the effects of concentration,
temperature, pH, etc. to characterize the
properties of the enzyme catalyzing the
reaction

10. Enzyme Kinetics
• An approach to understanding the
mechanism of action of enzymes
• An approach to understanding how
mutations may effect function
• An approach to understanding how
changes in the physical and chemical
environments change function

11. Rate Constant: k
• A B
• Velocity of Rx
– V=d[B]/dt
• d=change
– V=-d[A]/dt
• V=d[B]/dt = -d[A]/dt = k[A]
• Large k rapid Rx
• Small k slow Rx

12. The free energy of this reaction is not changed by the presence
of the enzyme, but for a ΔG<0.
A --------------> B
enzyme

13. The Transition State
Understand the difference between ΔG
and ΔG*
• The overall free energy change for a
reaction is related to the equilibrium
constant
• The free energy of activation for a reaction
is related to the rate constant
• It is extremely important to appreciate this
distinction!

15. 1) Velocity is dependent on [S].
2) The more enzyme added, the
faster the reaction goes.

16. Initial velocity Vo
• When enzyme is mixed with high
concentration of substrate [S] reaction goes
rapidly to steady state.
– Does not allow characterization
• Use low starting [S] and increase
• Hold [enzyme] constant
• Measure rate of reaction, Vo as [S] increases
– Until rate becomes constant: approaches Vmax

17. Effect of [Substrate]

18. A -------------> B
k1
A + B -----------> C
k2
d[A]/dt = k1[A]
For a first order reaction:
For a second order reaction:
The rate equation is:
The rate equation is:
d[A]/dt = k2[A][B]

19. E + S <=====> ES <=====> P + E
k2
k-2k-1
k1
In an enzymatic reaction:
But:
[ES] cannot be measured!
Nonetheless:
Initial [S] is known
[P] can be measured
[E]tot is known
Can we find [ES]?
Note: on initial conditions
[P] is negligibly small, So,
k-2[P]=0
Thus,
Vo=k2[ES] (1)

20. YES!
If we assume "steady state kinetic conditions". That is, [S] and [P] are
changing, but [ES] does not change (a constant flux of S "through" the
enzyme).
d[ES]/dt = 0 (1)
Also (from conservation of matter):
[E]tot = [E]free + [ES] (2)
Now, divide eq. 1 by [E]tot :
Vo/[E]tot = k2[ES]/[E]tot
Since d[ES]/dt = 0 the rate of formation of [ES] must equal the rate of
breakdown of [ES]
Vformation = k1[E]free[S] (2nd order rate equation)
Vbreakdown= k2[ES] + k-1[ES]
= (k2 + k-1)[ES] (Two 1st order rate equations)
k1[E]free[S] = (k2 + k-1)[ES] (Rates must be equal)
E + S <=====> ES <=====> P + E
k2
k-2k-1
k1

21. Rearranging, solving for [ES]:
[ES] = k1[E]free[S]/(k2 + k-1) (3)
If we define the Michaelis-Menten constant as Km = (k2 + k-1)/k1
and we substitute into eq. 3, we get:
[ES] = [E][S]/Km (4)
Now, let´s rearrange eq. 2 to [E]free = [E]tot - [ES] and then substitute into eq. 4
giving:
[ES] = ([E]tot - [ES])[S]/Km
Solving for [ES] gives:
[ES] = [E]tot(([S]/Km)/(1 + [S]/Km))
and then multiply top and bottom by Km, we get:
[ES] = [E]tot([S]/[S] + Km)

22. [ES] = [E]tot([S]/[S] + Km)
Finally, substitute into eq. 1 to get:
Vo = k2[E]tot([S]/[S] + Km) (5)
this is the expression for Vo in terms of known quantities, let’s try it!!

23. If [S] very large
[S] >> Km. The enzyme is saturated with substrate.
In this case, [S] + Km = [S], so eq. 5 becomes:
Vo = k2[E]tot([S]/[S]) or Vo = k2[E]tot
This is the rate at large substrate concentration, the maximal rate for
[E], which is called Vmax
Vmax = k2[E]tot
Substituting into eq. 5 gives the Michaelis-Menten Equation:
Vo = Vmax[S]/Km + [S] (6)
Note: This equation is derived from Vo, very little [P] has formed.
Vo = k2[E]tot([S]/[S] + Km)

24. If [S] is small:
[S] << Km Then the activity is in linear range.
[S] + Km = Km so eq. 6 becomes:
Vo = Vmax[S]/Km or Vo α [S]
Vo = k2[E]tot([S]/[S] + Km)
If [S] = Km
The definition of Km
Vo = Vmax[S]/[S] + [S] or
Vo = Vmax/2 = Km
Km is defined as the [S] that results in half maximal rate

25. Km and Vmax are
experimentally determined for
each enzyme using Vo vs. [S]
plots.
As V max is difficult to
determine (hyperbolic) the
Lineweaver-Burke plot is used.
Rearrange eq. 6 into linear form:
(1/Vo) = Km/Vmax(1/[S]) + 1/Vmax
So the plotted data looks like:

26. Understanding Km
The "kinetic activator constant"
• Km is a constant
• Km is a constant derived from rate constants
• Km is, under true Michaelis-Menten
conditions, an estimate of the dissociation
constant of E from S
• Small Km means tight binding; high Km
means weak binding

27. Understanding Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the
reaction - but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is asymptotically approached as
substrate is increased

28. Derivation of KCAT or turnover number:
For all catalyzed reactions:
[E]tot = [E]free + [ES] (2)
And with [S] >> Km:
Vmax = k2[E]tot or
k2 = Vmax/ [E]tot
For this reason k2 is also known as KCAT when the enzyme is saturated, so:
KCAT = Vmax/ [E]tot (7)
Therefore, when [S] is low, from eq. 7 and the Michaelis-Menten equation:
V = Vmax[S]/Km
then
V = KCAT/Km[S][E]
Therefore, KCAT/Km is a measure of how
rapidly and enzyme works and is referred as
the specificity constant or catalytic efficiency

29. The turnover number
A measure of catalytic activity
• kcat, the turnover number, is the number
of substrate molecules converted to
product per enzyme molecule per unit of
time, when E is saturated with substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec
to many millions per sec

30. The catalytic efficiency
Name for kcat/Km
• An estimate of "how perfect" the enzyme is
• kcat/Km is an apparent second-order rate
constant
• It measures how the enzyme performs
when S is low
• The upper limit for kcat/Km is the diffusion
limit - the rate at which E and S diffuse
together

32. Alcohol Dehydrogenase: ADH
CH3CH2OH + NAD+ CH3CH2O + H++ NADH
Catalyses conversion of ethanol to
aldehyde using co-enzyme NAD+
MH2 + NAD+ → NADH + H+ + M: + energy, where M is a
metabolite.
NAD+ oxidized to NADH reduced

33. NAD+ to NADH

34. NAD+

35. NADH

36. Reaction is complex
• ADH +ALC ADH-ALC
• ADH + NAD ADH-NAD
• ADH-NAD +ALC ADH-NAD-ALC
• We are not looking at this!

37. Alcohol Dehydrogenase
CH3CH2OH + NAD+ CH3CH2O + H+
+ NADH
We will measure the forward Rx (k 2)as increased absorbance
at 340. Only NADH absorbs at this wave length
Will find the assay conditions which produce max activity
Km

38. WHAT ARE WE MEASURING ?
• Production of NADH
– NAD+ NADH
– Wavelength shift
• Depends on participation of Alcohol and ADH
• How can you do this
• Ensure that NAD is not a rate limiting
component.
– [NAD] constant and high
– [ADH] constant
– [ETOH] low and increasing

39. Experiment
1.0
2.0
3.0
0
60 120 180 240 300 360 4200
time (seconds)
A 340
y = 0.0191 x - 1.0067
Kinetic curve
Add enzyme

40. Initial Velocity

41. Temperature Dependence

42. Effect of enzyme concentration
I
II
[E]std
[P]
time (min)
0 2 4 6 8 10

43. Effect of pH
V0
pH
pKa of reaction 1
~ 4.0
pKa of reaction 2
~ 9.0
2 124 6 8 10
max
low
Activity decreases due
to lysine deprotonation
Activity decreases due
to glutamate/aspartate
protonation
Maximal activity
range

45. ENZYME INHIBITORS

68. Enzyme Co-factors

69. APOENZYME APOENZYME APOENZYME
DEFINITION OF TERMS
Protein
part
Cofactor
(Non-protein
part)
Coenzyme
Prosthetic
group
Metal
ion
HOLOENZYME
+ ++

70. ENZYME COFACTORS
A. Coenzyme Enzyme
Chemical
Groups
Transferred
Vitamin
Precursor
Thiamine
Pyrophosphate
(TPP)
Pyruvate dehydrogenase,
Isocitrate dehydrogenase, α-
ketoglutarate dehydrogenase,
Transketolase, α-Ketoacid
dehydrogenase
Aldehydes Thiamine
(Vit B1)
Flavin Adenine
Dinucleotide
(FAD)
Succinate dehydrogenase, α-
Ketoglutarate dehydrogenase,
Pyruvate dehydrogenase,
Nitric oxide synthase
Electrons
Riboflavin
(Vit B2)
Nicotinamide
Adenine
Dinucleotide (NAD)
Lactate dehydrogenase;
Other dehydrogenases
Hydride ion
(:H-)
Nicotinic acid
(Niacin; B3)
Pyridoxal
Phosphate (PLP)
Glycogen phosphorylase,
γ-ALA synthase, Histidine
decardoxylase, Alanine
aminotransferase
Amino groups
Pyridoxine
(Vit B6)
Lipoate Pyruvate dehydrogenase
α-Ketoglutarate dehydrogenase
Electrons and
acyl groups
Not required
in diet

71. ENZYME COFACTORS
A. Coenzyme Enzyme
Chemical
Groups
Transferred
Vitamin
Precursor
Coenzyme A
(CoASH)
Acetyl CoA
carboxylase Acyl groups
Pantothenic
acid & other
compounds
Biocytin
Pyruvate
carboxylase,
Acetyl CoA
carboxylase,
Propionyl CoA
carboxylase
CO2
Biotin
5’-
deoxycobalamin
Methylmalonyl
mutase
H atoms and
alkyl groups
Vit B12
Tetrahydrofolalate
Thmidylate
synthase
One-carbon
groups
Folic acid

72. ENZYME COFACTORS: COENZYME A
C-CH2-CH2-N-C-C—C-CH2O
O
II
O
II
I
OH
H
II
O
I
CH3
CH3
I
I
NH
I
CH2
I
CH2
I
SH
O = P – O-
I
O
I
O = P –O-
I
O
NH2
N
N
N N
O
I
O = P – O-
I
O-
O
OH
Pantothenic acid
1. Pantothenic acid-derived,
co-factor of several
enzymes like acetyl CoA
carboxylase.
2. Takes part in reactions of
the CAC, FA synthesis and
oxidation, acylations and
cholesterol synthesis.
H
H
HH H
Active
sulfhydryl
group that form
thioesters with
acyl groups

73. ENZYME COFACTOR: NAD+
COO-
|
HO - C – H
|
CH3
COO-
I
C = O
I
CH3
Lactate
dehydrogenase
L-Lactate PyruvateNAD+ NADH+ + H+

74. COENZYME: BIOTIN
Pyruvate
Gluconeogenesis
Oxaloacetate
ATP ADP + Pi
Pyruvate
carboxylase
CO2BIOTIN
COO-
I
C =O
I
CH3
COO-
|
C= O
|
CH2
|
COO-

75. ROLE OF FAD AND FMN IN
NITRIC OXIDE (NO) SYNTHESIS
Arginine
NO +
NADPH2
+ O2
NADP+
+ H2O
Nitric oxide synthase
FAD, FMN, Heme
Tetrahydrobiopterin
COENZYME: FAD
NH2
I
H2N = C
I
NH
I
CH2
I
CH2
I
CH2
I
H3N – C – H
I
COO-
NH2
I
C = O
I
NH
I
CH2
I
CH2
I
CH2
I
H3N – C – H
I
COO-
+
Citrulline
+
+

76. Cofactor Enzyme
B. Inorganic (Metal ions
or iron- sulfur clusters)
Zn+2 Carbonic anhydrase, Alcohol dehydroge-
nase, Carboxypeptidases A & B
Cu+2 Cytochrome oxidase
Mn+2 Arginase, Ribonucleotide reductase
Mg+2 Hexokinase, Pyruvate kinase, Glucose 6-
phosphatase
Ni+2 Urease
Mo Nitrate reductase
Se Glutathione peroxidase
Mn+2 Superoxide dismutase
K+ Propionyl CoA carboxylase
ENZYME COFACTORS

77. METALLOENZYMES
Enzymes that require
a metal in their
composition

78. ENZYME COFACTORS: Mg+2
Glucose Glucose 6-PO4
Hexokinase
Glucokinase
ATP ADP
+ Pi
Mg+2
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 – OH
|
H
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O – P
|
H

79. GLYCOLYSIS
COO-
|
C2 = O
|
CH3
O
||
C1 – O-
|
C2 – O ~ P
|
H – C3
|
H
Phosphoenol Pyruvate
(PEP)
Pyruvate
ADP ATP
K+
Δ G0 = - 6.1 kcal/mole
ENZYME COFACTORS: K+
Pyruvate kinase

80. ENZYME COFACTORS: Zn+
CO2 + H2O H2CO3
Carbonic anhydrase
Zn+2

81. Hexokinase/
Glucokinase
ATP ADP
Δ G0 = - 4.0 kcal/mole
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 – OH
|
H
Glucose
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O – P
|
H
Glucose 6-Phosphate
ATP AS A CO-SUBSTRATE
Mg+2

82. LYSOZYME: ACTIVE SITE

83. CHYMOTRYPSIN:ACTIVE SITE

84. REGULATORY ENZYME
The enzyme that catalyzes the
rate-limiting or committed
step of a metabolic
pathway.

85. ALLOSTERIC MODIFICATION:
Phosphofructokinase I

86. REGULATORY ENZYME
Phosphofructokinase I
Fructose 6-
phosphate
Fructose 1,6-
bisphosphate
ATP ADP + Pi
Phosphofructokinase I
Glycolysis
H
|
H - C1 - OH
|
C2 = O
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O – P
|
H
H
|
H - C1 - O - P
|
C2 = O
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O – P
|
H
AMP
F 2,6 bisPO4
ATP
Citrate
H+
+ -

87. ISOENZYME
 Different structural forms of an
enzyme which catalyze the same
chemical reactions → act on the
same substrate(s) and produce the
same product(s) but exhibit differing
degrees of efficiency.
 Different isoenzymes are expressed in
specific tissues of the body.

88. ISOENZYMES OF LACTATE
DEHYDROGENASE
Lactate dehydrogenase
(LDH) – catalyzes the
reversible conversion of
pyruvate to lactate.
Tetramer consisting of 2
subunits: M (found in
skeletal muscles and
liver) & H (heart).
5 distinct isoenzyme forms
(from combination of M &
H isozymes).
An increase of H4 in the blood
indicates tissue damage
as in heart attack.
Enzymes can therefore serve
as markers for disease.

89. SIX MAJOR CLASSES OF ENZYMES (IUBMB*, 1964)
CLASS EXAMPLE
Oxidoreductases Dehydrogenases, Oxidases, Reductases,
Peroxidases, Catalases, Oxygenases,
Hydroxylases
Transferases Transaldolase and Transketolase; Acyl, methyl
glucosyl, and phosphoryltransferases,
Kinases, Phosphomutases, Transaminases
Hydrolases Esterases, Glycosidases, Peptidases,
Phosphatases, Thiolases, Phospholipases,
Amidases, Deaminases, Ribonucleases
Lyases Decarboxylases, Aldolases, Hydratases,
Dehydratases, Synthases, Lyases
Isomerases Epimerases, Isomerases, Mutases, Racemases
Ligases Synthetases, Carboxylases
*International Union of Biochemistry and Molecular Biology; classification is
based on the reactions enzymes catalyze; each class is divided into subclasses.

90. OXIDOREDUCTASES
Transfer of electrons and hydrogen
atoms from donors (or reductants,
hence oxidized to acceptors (or
oxidants, hence reduced).
COO-
|
HO – C – H + NAD+
|
CH3
L-Lactate
COO-
|
C = O + NADH + H+
|
CH3
Pyruvate
Lactate
dehydrogenase

91. TRANSFERASES
Transfer functional groups (like C-, N-, or
P-) from donors to acceptors; utilize 2
substrates to produce 2 products.
COO-
|
H3N – C – H + C = O
| |
CH3 (CH2)2
L-Alanine |
COO-
α-Ketoglutarate
(keto acid)
COO- COO-
| |
C = O + H3N – C – O
| |
CH3 (CH2)2
Pyruvate |
COO-
L-Glutamate
(amino acid)
Alanine
transaminase
PLP
(amino acid) (keto acid)
substrate
substrate
product
product

92. HYDROLASES
Catalyze cleavage of chemical bonds by
addition of H2O, producing 2 products
O O
|| ||
-O – P ~ O – P ~ O- + HOH
| |
-O O-
Pyrophosphate
(PPi)
O
||
2 HO – P – O-
|
-O
Phosphate
2 (Pi)
Pyrophosphatase
Phosphate bond

93. LYASES
Cleave C-C, C-O, C-N bonds by means
other than hydrolysis or oxidation
O O-
/
C
|
C = O H+
|
CH3
Pyruvate
H O
//
C + O = C = O
| Carbon
CH3 dioxide
(CO2)
Pyruvate
decarboxylase
Acetaldehyde

94. ISOMERASES
Transfer of functional groups or double
bonds within the same molecule
C1OO-
|
H3N – C2 – H
|
C3H3
L-Alanine
C1OO-
|
H – C2 – NH3
|
C3H3
D-Alanine
Alanine
racemase

95. CH3
|
C = O
|
COO-
COO-
|
CH2
|
C = O
|
COO-Biotin-
CO2
ATP ADP + Pi
Oxaloacetate
Pyruvate
Pyruvate carboxylase
ATP ADP
+ Pi
LIGASES
Catalyze the joining of
substrates in the presence of ATP.

96. REGULATION

97. REGULATION OF ENZYME
ACTIVITY
Feedback Inhibition
Allosteric (Non-covalent)
Modification
Covalent Modification
Zymogen Activation
Induction or Repression
of Enzyme Synthesis

98. 98
Saturation curve of allosteric enzymes is sigmoidal
[S]
vo aktivace
inhibicebez
efektoru
activation
without
effector
inhibition

99. Cooperative effect
• in oligomeric enzymes and non-catalyzing proteins (e.g.
Hb)
• more subunits = more active sites
• binding substrate (or O2 to Hb) to one subunit/active site
induces conformation changes in other subunits/active
sites so that other substrate (or O2) molecules bind more
easily (or more hardly)
• example: hemoglobin (tetramer) × myoglobin (monomer)

100. large and complex
having quaternary structures
Allosteric Enzymes are:
Few enzymes:
single
subunit
Three
subunits
Four
subunits
Allosteric enzymes

101. What are the subunits called?
Catalytic subunit:
has the active site
on it
Regulatory subunits:
has the allosteric site
bind inhibitors &
activators

102. ‘Allostery’ means ‘different
shape’ [refers to the two shapes
of the enzyme]
Allosteric enzymes have TWO sites:
E
Active site
Allosteric site
Substrate
cannot fit into
the active site
Inhibitor
molecule
Inhibitor fits into
allosteric site
E

103. Allosteric enzymes are regulatory
enzymes that have two structurally
distinct forms: ACTIVE & INACTIVE
are regulatory enzymes that have two
structurally distinct forms:
are regulatory enzymes that have two
structurally distinct forms:

104. The binding of an:
Inhibitor & activator = effectors
Activator:
stabilizes the
active form of the
enzyme
Inhibitor:
stabilizes the
inactive form of the
enzyme

105. Allosteric inhibition
is a type of
reversible
inhibition which
allows the rate of
enzyme catalysed
reactions to be
controlled

106. PFK is a quaternary protein and has two allosteric
regulatory sites and a catalytic site.
Phosphofructokinase (PFK)
 is an allosteric enzyme
 regulates rate of
respiration

107. ATP: allosteric inhibitor of PFK
AMP/ADP: allosteric activators of PFK

108. Fructose-6-phosphate
PFK
Fructose-1,6-bisphosphate

109. PFK controls rate of respiration

110. End-product
regulation of a
pathway:
a case of allosteric
inhibition

111. FEEDBACK INHIBITION
Original Precursor(s)
Enzyme 1
Enzyme 2
Enzyme 3
Enzyme 4
Enzyme 5
1
2
3
Final Products

112. FEEDBACK INHIBITION
Carbamoyl PO4 + Aspartate
Carbamoyl aspartate
Aspartate transcarbamoylase
(ATCase)
series of
reactions
Cytidine triphosphate (CTP)
RNA & DNA
synthesis

113. FEEDBACK INHIBITION OF HMG CoA
REDUCTASE BY CHOLESTEROL
Acetyl
CoA
Acetyl
CoA
HMG CoA
Mevalonic acid + CoA
HMG CoA
reductase
Cholesterol
Feedback
inhibition
Acetoacetyl
CoA
> 25 steps

114. ALLOSTERIC MODIFICATION
Allosteric modulator (activator or inhibitor)
Binds to regulatory or allosteric site
Conformational change in the
regulatory enzyme
Effect is transmitted to the active site
Change in shape of the active site
Altered activity

115. The end-product of the reaction:
can act as a non-competitive inhibitor and
bind at a site on the enzyme

116. Negative Feedback
• In feedback inhibition, the end product of
a metabolic pathway shuts down the
pathway

117. What is the value of negative
feedback inhibition to a cell?
Prevents a cell from wasting
chemical resources by
synthesizing more product than
is needed

118. COVALENT MODIFICATION
ATP ADP
ENZYME- Ser -- OH
HPO4
= H2O
Protein
kinase
Phospho-
protein
phosphatase
ENZYME- Ser – O – PO3
2-

119. COVALENT MODIFICATION
OF THE ENZYME
Glycogen phosphorylase
AMP
ATP
and/or
G6P
Glucose
2 ATP 2 ADP
2 H2O2 P
Phosphorylase
kinase
Phosphorylase b
(inactive)
Phosphorylase a
(active)
P
P
P P
Phosphoprotein
phosphatase

120. COVALENT MODIFICATION: PYRUVATE
DEHYDROGENASE
Pyruvate
dehydrogenase
Pyruvate
dehydrogenase
P
Pyruvate
dehydrogenase
kinase
ATP ADP
Pyruvate
dehydrogenase
phosphatasePi H2O
(inactive)(active)

121. ENZYMES Low activity High activity
Acetyl CoA Carboxylase EP E
Glycogen synthase EP E
Pyruvate dehydrogenase EP E
HMG CoA reductase EP E
Glycogen phosphorylase E EP
Citrate lyase E EP
Phosphorylase b kinase E EP
HMG CoA reductase kinase E EP
MAMMALIAN ENZYMES WHOSE CATALYTIC ACTIVITY IS ALTERED
BY COVALENT PHOSPHORYLATION-DEPHOSPHORYLATION
E = Dephosphorylated EP = Phosphoenzyme

122. ZYMOGEN ACTIVATION:
BLOOD COAGULATION
Clotting Factors
Prothrombin Thrombin
Fibrinogen Fibrin
Ca+2
Some of the processes
involved in blood clotting

123. INDUCTION OR REPRESSION
OF ENZYME SYNTHESIS
↑ Blood glucose
levels
(Well-fed state)
↑ Insulin
↑ Synthesis of
key enzymes involved
in glucose degradation
↓ Blood glucose levels
(Starvation)
↑ Glucagon
↑ Synthesis of
key enzymes involved
in glucose synthesis

124. Three utilizations of enzymes in medicine
1. enzymes as indicators of pathological condition
2. enzymes as analytic reagents in clinical chemistry
3. enzymes as drugs

125. 125
Examples of enzymes in clinical diagnostics
Enzyme Reference values Elevation in serum indicates
ALT
CK
PSA
up to 0,9 kat/l
up to 4 kat/l
up to 4 μg/l
hepatopaties
myopaties, myocardial
infarction
prostate cancer
ALT alanine aminotransferase, CK creatine kinase,
PSA prostate specific antigen
In cell damages, activity of intracellular enzymes in
extracellular fluid (blood serum) is elevated

126. Creatine kinase (CK) is a dimer and makes three isoenzymes
Isoenzyme Organ
% of total
activity
in blood plasma
Elevated value
CK-MM
CK-MB
CK-BB
muscles
heart
brain
94-96 %
up to 6 %
traces
muscle trauma
infarction
brain damage
• catalyze the same reaction
• differ in primary structure, physical, and kinetic properties
• often have different tissue distribution
• determined mostly by electrophoresis and/or by immunochemical assays
Isoenzymes/Isoforms

127. Enzymes as analytic reagents
Enzyme Enzyme origin Assay for
Glucose oxidase
Peroxidase
Lipase
Cholesterol oxidase
Uricase
Bilirubin oxidase
Urease
Lactate
dehydrogenase
Taq polymerase
Aspergillus niger
horse radish (Armoracia sp.)
Candida sp.
Pseudomonas sp.
Candida sp.
Myrothecium sp.
Canavalia sp.
Pediocus sp.
Thermus aquaticus
glucose
glucose
triacylglycerols
cholesterol
uric acid
bilirubin
urea
ALT, AST
PCR method

128. Enzymatic determination of glucose
glucose + O2  gluconolactone + H2O2
H2O2 + H2A  2 H2O + A
glucose
oxidase
peroxidase
colourless
chromogen
coloured product
(absorbance measured)
The principle of glucose determination in biochemical
analyzers and in personal glucometers

129. 129
Pancreatic enzymes in
therapy
• enzyme mixtures (lipase, amylase,
proteinases) of animal origin
• indication: insufficient secretion of
pancreas, cystic fibrosis
• 3 × times daily a capsule during meal
acidoresistant capsules, they survive the
passage through stomach, soluble till in
duodenum

130. Asparaginase in leukemia treatment
• Catalyzes the hydrolysis of asparagine amide group (deamidation)
• Asn + H2O  Asp + NH3
• L-asparagine is necessary for the proteosynthesis of some cancer
cells
• Hydrolysis of Asp reduces the cell proliferation (see also Seminars, p.
19)
Enzyme fibrinolytics
• thrombolytic drugs, dissolve blood clots in veins
• urokinase (human enzyme, serine protease)
• converts plasminogen to plasmin  which degrades fibrin  thrombolysis
• venous thrombosis, pulmonary embolism, acute myocardial infarction

131. Proteases in enzyme therapy
Local treatment
• fibrinolysin, chymotrypsin, collagenase and other
• degrade necrotic tissue, clean wounds, decubital ulcers etc.
Systemic treatment
• trypsin, chymotrypsin, papain (papaya), bromelain (pineapple)
• anti-inflammatory agents
• sports injury, trauma, arthritis, other kinds of swelling, arthritis etc.
• Wobenzym, Phlogenzym and other