Globular proteins are spherical proteins that contain heme as a prosthetic group. Globular heme proteins function as electron carriers, parts of enzyme active sites, and carriers of oxygen and carbon dioxide like hemoglobin and myoglobin. Myoglobin stores oxygen in muscle cells and facilitates oxygen transport between hemoglobin and cells. Hemoglobin transports oxygen in red blood cells through a cooperative binding mechanism between its four heme groups that allows for high oxygen affinity when oxygen levels are high. Both proteins bind oxygen reversibly to their heme groups through interactions with proximal and distal histidine residues.

1. Globular Proteins

2. Globular proteins are characterized as generally
having:
• a variety of different kinds of secondary structure
• spherical shape
• good water solubility
• a catalytic/regulatory/transport role i.e. a dynamic
metabolic function

3. Globular heme proteins contain heme as prosthetic
group.
Functions of globular hemeproteins include:
• electron carriers
• part of enzyme active site
• transport of O2 and CO2- hemoglobin
• storage of O2-myoglobin

4. • II.
Globular
Hemeproteins
• Contain
heme
as
prosthe.c
group
• Role
of
heme
is
dependent
on
environment
created
by
3D
structure
of
protein
• Heme
of
cytochrome
→
electron
carrier
• Heme
of
catalase
→
part
of
ac.ve
site
• Heme
of
Hb
and
myoglobin
→
binds
O2
reversibly

5. • A.
Structure
of
Heme
• Complex
of
Protoporphyrin
IX
&
Fe2+
• Fe2+
bound
to
4
Ns,
other
2
bonds
perpendicular
to
plane
of
ring
available
for
bonding
• In
Hb,
one
of
these
aHached
to
N
terminus
of
His,
other
binds
O2.

6. Structure of heme
porphyrin heme (Fe-protoporphyrin IX)

7. heme
“proximal”
histidine
“distal”
histidine

8. B.
Structure
and
func9on
of
myoglobin
• It
is
a
heme
protein
present
in
heart
and
skeletal
muscle
• Reservoir
for
O2
and
carrier
of
O2
in
muscle
cell
• Single
polypep.de
chain
similar
to
polypep.des
in
Hb
• 1.
α-­‐helical
content:
• ~
80%
of
pep.de
in
8
stretches
of
α-­‐helix
Labeled
A
to
H
• Terminated
by
Pro
or
β-­‐bends
and
loops
stabilized
by
H
bonds
and
ionic
bonds.

9. • 2.
Loca9on
of
polar
and
nonpolar
amino
acid
residues:
• Interior
made
up
of
hydrophobic
amino
acids
stabilized
by
hydrophobic
interac.ons
• Surface
→
charged
amino
acids
–
form
H
bonds
with
water
• 3.
Binding
of
heme
group:
• Heme
in
crevice
lined
with
non-­‐polar
amino
acids,
except
2
His
residues
• Proximal
his9dine
–
binds
directly
to
Fe2+
of
heme
• Distal
his9dine
stabilizes
binding
of
O2
to
Fe2+

12. O2 Binding in Mb and
Hb

14.
C.
Structure
and
func9on
of
hemoglobin
• Found
exclusively
in
RBCs
→
transports
O2
• Hb
A
–
predominant
form
in
adults:
4
polypep.de
chains
-­‐-­‐
α2β2
• Each
subunit
–
heme-­‐binding
pocket
similar
to
myoglobin
• Can
transport
O2
and
CO2
• O2-­‐binding
proper.es
affected
by
allosteric
effectors,
unlike
myoglobin

15. 1.
Quaternary
structure
of
hemoglobin:
• 2
iden.cal
dimers:
(αβ)1
and
(αβ)2
• dimers
held
together
by
hydrophobic
interac.ons
(on
contact
surfaces
of
subunits
as
well
as
internally)
but
ionic
and
H-­‐bonding
also
exist
• 2
dimers
held
together
by
weak
polar
bonds
• different
conforma.on
in
deoxyHb
and
oxyHb

16. αβ dimer 2
αβ dimer1

17. T and R forms of Hemoglobin
T = “taut” → deoxy Hb → low affinity for O2
R = “relaxed” → oxy Hb → high affinity for O2

18. • a.
T
form:
“taut”
form
• deoxy
form
of
Hb
• 2
αβ
dimers
joined
by
ionic
and
H-­‐bonds
• low
oxygen-­‐affinity
form
of
Hb
• b.
R
form:
• binding
of
O2
disrupts
some
ionic
and
H-­‐
bonds
between
αβ
dimers
• “relaxed”
form
• high
oxygen-­‐affinity
form
of
Hb

19.
D.
Binding
of
oxygen
to
myoglobin
and
hemoglobin
• D.
Binding
of
oxygen
to
myoglobin
and
hemoglobin
• Myoglobin
→
one
heme
→
binds
one
O2
• Hb
→
4
heme→
binds
4
O2
• Hb
binding:
degree
of
satura.on
(Y)
from
0
to
100%
• 1.
Oxygen
dissocia9on
curve:
• plot
of
Y
against
PO2
• myoglobin
:
higher
affinity
for
O2
than
Hb
• P50
is
1
mm
Hg
for
myoglobin
and
26
mm
Hg
for
Hb

21. • a.
Myoglobin:
• O2
dissocia.on
curve
hyperbolic
• This
reflects
that
myo
binds
single
O2
• Mb
+
O2
MbO2
they
exist
in
equilibrium
• Exchange
between
Hb
and
Mb,
Mb
and
muscle
cells
depending
on
equilibrium
• Mb
binds
O2
released
from
Hb,
releases
when
O2
drops.
Mb
then
releases
the
O2
into
the
muscle
cell.
This
only
happens
when
there
is
an
O2
demand.

22. • b.
Hemoglobin:
• O2
dissocia.on
curve
is
sigmoidal
• Coopera.ve
bind
of
O2
(increased
affinity
for
Hb
with
more
binding)
• Heme-­‐heme
interac.on:
binding
of
O2
at
one
heme
increases
affinity
for
O2
at
others

24. • E.
Allosteric
effects
• Ability
of
Hb
to
bind
O2
depends
on
allosteric
(“other
site”)
effectors:
– PO2
– pH
of
environment
– PCO2-­‐
an
inc
will
cause
the
inc
in
unloading
of
O2.
– 2,3-­‐disphosphoglycerate
availability
• allosteric
factors
do
not
affect
myoglobin

25. • 1.
Heme-­‐heme
interac9ons:
• structural
changes
in
one
heme
group
transmiHed
to
others
• affinity
for
last
O2
~300X
affinity
for
first
O2
• a.
Loading
and
unloading
of
oxygen:
• more
O2
can
be
delivered
to
.ssues
with
small
changes
in
PO2
• Graph
showing
loading
and
unloading
at
different
par.al
pressures
of
O2.
Hb
alterna.vely
carries
O2
and
CO2
between
lungs
and
.ssues
• b.
Significance
of
sigmoidal
O2-­‐dissocia9on
curve
Compare
a
hyperbolic
curve
to
a
sigmoidal
curve
• A
sigmoidal
curve
gives
increasing
affinity
of
O2
for
Hb
with
increasing
par.al
pressure
while
a
hyperbolic
curve
is
a
straight
line
in
that
range.

27. • 2.
Binding
of
CO2:
• Most
of
the
CO2
in
the
blood
is
transported
as
bicarbonate:
• CO2
+
H2O
H2CO3
• H2CO3
HCO3-­‐
+
H+
• Some
CO2
binds
to
the
terminal
–NH2
of
the
α
and
β
chains
forming
carbaminoHb.
•
Binding
of
CO2
stabilizes
the
“taut”
form
of
Hb
(deoxyHb).

28. • 3.
Binding
of
CO:
• CO
binds
reversibly
to
the
Fe2+
the
same
way
that
O2
does
• CO
+
Hb
HbCO
(carbon
monoxy
Hb)
• Affinity
of
Hb
for
CO
is
220X
affinity
for
O2
• Binding
of
CO
to
Hb
increases
affinity
of
remaining
sites
for
O2
• O2
dissocia.on
curve
shigs
to
leg
(becomes
hyperbolic)
• >
60%
HbCO
fatal
• treated
with
O2
therapy

29. 4.
Bohr
Effect:
• Shig
of
O2
dissocia.on
curve
to
the
right
with
decrease
in
pH
or
increase
in
PCO2
• This
translates
to
a
decreased
affinity
of
Hb
for
O2
under
these
condi.ons,
therefore
you
unload
O2
easier

30. • a.
Source
of
the
protons
that
lower
the
pH:
• 2
principle
sources
of
protons:
– lac.c
acid
produced
by
anaerobic
metabolism
in
muscles
– increased
produc.on
of
CO2
by
cell
u.liza.on
of
O2
through
respira.on:
• CO2
+
H2O
H2CO3
H+
+
HCO3-­‐
– in
lungs
the
equilibrium
of
this
reac.on
is
towards
the
leg
because
CO2
is
lost
through
exhaling
• the
decreased
affinity
of
Hb
for
O2
under
the
Bohr
effect
condi.ons
results
is
greater
off
loading
(release)
of
O2
in
the
.ssues.

31. The Effect of CO2 and H+ on O2 Binding
Bohr Effect:
Increased concentrations of CO2 and H+ promote
the release of O2 from hemoglobin in the blood.

32. How do CO2 and H+ promote the release of O2
from hemoglobin?
• presence of “salt bridge” • no ionic interaction in
in T form R form

33. CO2 is bound to hemoglobin at protein interfaces, not
Fe2+ center!

34. • Summary
reac.on
for
the
Bohr
effect:
• HbO2
+
H+
HbH+
+
O2
OxyHb
DeoxyHb
• Equilibrium
shigs
to
the
right
when
H+
conc.
increases
(decrease
in
pH),
while
it
shigs
to
leg
when
PO2
increases.
• The
protonated
forms
of
the
terminal
α-­‐
subunit
–NH2
groups
and
His
side-­‐chains
stabilize
the
T
form
(deoxy
form)
of
Hb.

35. • 5.
Effect
of
2,
3-­‐bis-­‐
phosphoglycerate(BPG)
on
oxygen
affinity:
• Important
regulator
of
Hb
binding
O2
• Most
abundant
organic
phosphate
in
RBC
(conc.
~
=
conc.
of
Hb)
• Synthesized
from
intermediate
of
glycolysis
• a.
Binding
of
2,3-­‐BPG
to
deoxyhemoglobin:
• Binds
to
deoxyHb
stabilizing
it
• Decreases
affinity
of
Hb
for
O2

36. • b.
Binding
site
of
2,3-­‐BPG:
• 1
molecule
of
2,3-­‐BPG
binds
to
a
pocket
between
the
β-­‐chains
in
the
center
of
the
deoxyHb
center
• expelled
on
oxida.on
of
Hb
(pocket
disappears)
• c.
ShiX
of
oxygen-­‐dissocia9on
curve:
• Blood
stripped
of
2,3-­‐BPG
has
a
high
affinity
for
O2
• 2,3-­‐BPG
shigs
the
O2-­‐dissocia.on
curve
to
the
right
allowing
decreased
affinity
of
Hb
for
O2
and
effec.ve
unloading
of
O2
in
.ssues
• similar
to
Bohr
effect
but
no
difference
between
lungs
and
.ssues

38. • d.
Response
of
2,3-­‐BPG
levels
to
chronic
hypoxia
or
anemia:
• 2,3-­‐BPG
increases
in
chronic
hypoxia
• chronic
hypoxia
can
be
caused
by
– pulmonary
emphysema
or
– high
al.tudes
or
– chronic
anemia
• increased
2,3-­‐BPG
shigs
O2
dissocia.on
further
to
right
allowing
greater
unloading
of
O2

40. • e.
Role
of
2,3-­‐BPG
in
transfused
blood:
• 2,3-­‐BPG
essen.al
for
normal
transport
func.on
of
blood
• Without
normal
concs.
of
2,3-­‐BPG,
Hb
becomes
an
O2
trap
(doesn’t
unload;
high
affinity)
• Blood
for
transfusion
formerly
stored
in
acid-­‐citrate-­‐
dextrose
medium
decreased
2,3-­‐BPG
conc.
→
“stripped”
blood
• Body
restores
conc.
of
2,3-­‐BPG
in
24
–
48
h
• 2,3-­‐BPG
can
be
restored
by
adding
inosin

41. Minor Hemoglobins

42. Minor Hemoglobins

43. Minor Hemoglobins
Embryonic form is Hb Gower 1
(ζ2ε2) (yolk sac).
HbF - 2 α chains, 2 γ chains (β-
chain family) - major form in
fetus and newborn (fetal liver –
2 weeks).
HbA - 2 β chains, 2 α chains -
major form in adult.
Fetal bone marrow begins
synthesizing HbA around 8th
month.

44. Globin gene organization

45. Steps in globin chain synthesis:
1. Transcription
2. Modification of mRNA precursor
by splicing
3. Translation by ribosomes &
further modifications (i.e.
glycosylation)

46. Hemoglobinopathies
• caused by abnormal structure of Hb
• characterized by low levels of normal Hb
Sickle-cell anemia (Hemoglobin S disease)
Hemoglobin C disease
Hemoglobin SC disease
Thalassemias – α thalassemia
β thalassemia

47. Sickle-cell anemia (HbS disease)
• abnormal β chain. HbS = α2βS2
• β chain mutation - glu 6 à val 6
• glu is negatively charged, val is nonpolar.
• only has effect postnatally because HbF is major
species in fetus
• symptoms - hemolytic anemia, painful crises,
poor circulation, frequent infections
• heterozygotes - HbA and HbS both present - 1 in
10 African Americans; "sickle cell trait" - no
symptoms, normal life span

48. Sickle-cell anemia (HbS disease)
• glutamic acid is replaced by valine at position 6 of β
chain

50. normal RBCs sickled RBCs

51. Symptoms worsen when Hb is in deoxy form - decreased pO2,
increased CO2, decreased pH, increased 2,3-BPG

52. Low solubility of HbS
causes aggregation and
distortion of cell shape.

53. HbS
• val instead of glu at
position 6
HbA
• glu at position 6
HbC
• lys instead of glu at
position 6
HbSC
• HbS as well as HbC
present → 2 bands in
electrophoresis

54. HbC disease
• lys instead of glu at position 6
• HbC homozygotes - mild, chronic hemolytic anemia. Not life-
threatening
HbSC disease
• HbS as well as HbC present → 2 bands in electrophresis
• usually undiagnosed until infarctive crisis occurs (childbirth,
surgery)
• can be fatal

55. Thalassemias
• hereditary hemolytic diseases
• most common genetic disorder in humans
• heterogeneous collection of diseases

56. β-thalassemias
• synthesis of β-chain decreased or absent
β-thalassemia minor (or trait) - one normal, one defective β-
chain gene. Not life-threatening
β-thalassemia major - both genes defective. Normal at birth.
Severe anemia by age 1-2.
Treatment requires frequent transfusions → Leads to iron
overload (hemosiderosis).
Death between 15-25 years old. Bone marrow transplant
(BMT) is an option.

58. α-thalassemias
• decreased or absent α chain synthesis
• severity of disease depends upon the number
of defective α genes:
0 defective - normal
1 defective - silent carrier of α-thalassemia. No
symptoms
2 defective - α-thalassemia trait - no serious
symptoms
3 defective - Hemoglobin H disease - moderately
severe hemolytic anemia
all 4 defective - hydrops fetalis - fetal death (α
chains needed for HbF)

59. Methemoglobinemia
• 1.
Forma9on
of
methemoglobin
• Oxida.on
of
Fe2+
→
Fe3+
converts
Hb
and
myoglobin
to
metHb
and
metmyoglobin
• Cannot
bind
O2,
• Oxida.on
by
drugs
like
nitrates,
H2O2
or
free
radicals
or
muta.on
in
α-­‐
or
β-­‐chain
of
globin
→
methemoglobinopathy
(HbM).
• a.
Reduc9on
of
methemoglobin:
• Normal
oxida.on
corrected
by
NADH-­‐cytochrome
b5-­‐
reductase
• RBCs
of
newborns
→
half
the
capacity
of
this
enzyme,
therefore
more
suscep.ble
to
oxida.on

60. Fibrous Proteins

61. Fibrous
proteins
are
characterized
as
generally
having:
•
one
domina.ng
kind
of
secondary
structure
(i.e.
collagen
helix
in
collagen)
•
a
long
narrow
rod-­‐like
structure
•
low
water
solubility
•
a
role
in
determining
.ssue/cellular
structure
and
func.on
(e.g.
collagen,
α-kera.n)

62. Collagen
-­‐
most
abundant
protein
in
body;
rigid,
insoluble
Elas.n
-­‐
stretchy,
rubber-­‐like,
lungs,
walls
of
large
blood
vessels,
ligaments

63. Structure
of
Collagen
Tropocollagen
is
a
right-­‐handed
triple
helix
formed
of
α-­‐chains.

64. Structure
of
Collagen
The
α-­‐chains
(individual
polypep.des
composing
tropocollagen)
consist
of
-­‐[Gly-­‐X-­‐Y]-­‐
repeats.
Proline
and
hydroxyproline/hydroxylysine
are
ogen
present
in
the
X
and
Y
posi.ons,
respec.vely.

67. Synthesis
of
collagen
•
made
in
fibroblast,
osteoblasts
(bone),
chondroblasts
(car.lage)
•
secreted
into
ECM
•
enzyma.cally
modified
•
aggregate
and
are
cross-­‐linked

68. Structure
of
tropocollagen
molecule

69. Biosynthesis
of
collagen
1. forma.on
of
pro-­‐α-­‐chains
-­‐
contains
signal
sequence
–
promotes
binding
of
polysome
to
RER
and
secre.on
into
the
cisternae;
signal
sequence
removed
2. some
pro
and
lys
residues
(in
the
Y
posi.on
of
gly-­‐X-­‐Y)
are
hydroxylated
by
prolyl
hydroxylase
and
lysyl
hydroxylase;
needs
molecular
O2
and
reducing
agent
like
ascorbic
acid
(from
vitamin
C).
3. glycosyla.on
-­‐
glucose
and
galactose
added
to
hydroxylysines;
pro-­‐α-­‐chains
join
to
form
procollagen.
N-­‐
and
C-­‐terminal
extensions
form
interchain
disulfide
bonds;
central
triple
helix
formed
because
of
favorable
alignment;
Transported
to
Golgi,
packaged,
and
secreted
as
procollagen.

70. Biosynthesis
of
collagen

71. Biosynthesis
of
collagen
(cont’d)
4.
N-­‐procollagen
pep.dase
and
C-­‐procollagen
pep.dase
remove
terminal
extensions,
leaving
triple
helical
collagen
(occurs
extracellularly).
5.
collagen
fibrils
-­‐
form
by
associa.on
of
collagen
molecules
with
about
a
3/4
overlap
with
other
molecules
(staggered,
parallel
arrays)
5.
cross-­‐linking
-­‐
interchain
cross-­‐links
caused
by
lysyl
oxidase
(a
pyridoxal
phosphate
and
copper-­‐requiring
enzyme);
O2
required;
oxida.ve
deamina.on
of
lysines
and
hydroxylysines;
Allysine
(aldehyde)
reacts
with
amino
group
of
nearby
lysine
or
hydroxylysine
to
form
interchain
cross-­‐
link.
Very
important
for
tensile
strength
of
collagen.

73. Ascorbate
coenzyme
required
by
prolyl/lysyl
hydroxylase
in
hydroxyla.on
step.
Vitamin
C
(ascorbate)
deficiency
results
in
scurvy
(collagen
can’t
be
cross-­‐
linked).

74. Cross
links
formed
by
lysyl/ Cu2+/
prolyl
oxidase
vitamin
B6
-­‐
copper
coenzyme
Number
of
cross-­‐links
increases
with
age
→
causes
s.ffening,
decreased
elas.city
of
skin
and
joints.

75. Biosynthesis
of
collagen
(con’t)
In
the
final
step,
collagen
fibrils
form
spontaneously
from
tropocollagen.
covalent
X-­‐links
between
Allysine
and
hydroxylysine
tropocollagen
molecule
triple
helix
of
α-­‐chains.

76. Types
of
Collagen
Common
Type Representative Tissues
disorders
Ehlers-Danlos
Osteogenesis
I Imperfecta
skin, bone, tendons, cornea
Marfan’s
cartilage, intervertebral disks, vitreous
II -
body
blood vessels, lymph nodes, dermis,
III Ehlers-Danlos
early phases of wound repair
Alport’s
IV basement membranes
Goodpasture’s
X - epiphyseal plates

77. Collagen
Degrada.on
and
Disorders
•
degrada.on
of
collagen
by
collagenase
allows
remodeling
of
ECM
Ehlers-­‐Danlos
–
hyperextensive
joints,
hyperelas.city
of
skin,
aor.c
aneurisms,
rupture
of
colon,
skin
hemmorhages
due
to
muta.on
in
α-­‐chains
Osteogenesis
Imperfecta
–
briHle
bone
disease,
mul.ple
fractures,
blue
sclera,
hearing
loss,
retarded
wound
healing

78. Ehlers-­‐Danlos
Syndrome
Hyperextension
of
skin

79. Osteogenesis
Imperfecta
(Blue
sclera)

81. In
Utero
Radiograph:
•
crumpled
long
bones
•
beaded
ribs

83. Elas.n
•
rubber-­‐like
proper.es
•
connec.ve
.ssue
protein
•
lungs,
large
blood
vessels,
elas.c
ligaments
Composi.on:
-­‐
small
nonpolar
amino
acids
(Gly,
Ala,
Val)
-­‐
also
rich
in
Pro
and
Lys
-­‐
liHle
or
no
OH-­‐Pro
or
OH-­‐Lys

84. Elas.n

85. Elas.n
•
3D
network
of
cross-­‐linked
polypep.des
•
cross
links
involve
Lys
and
alLys
•
4
Lys
can
be
cross-­‐linked
into
desmosine
•
desmosines
account
for
elas.c
proper.es

86. Elas.n
Degrada.on
and
Disorders
•
in
lungs
-­‐
lung
alveolar
elas.n
in
constantly
exposed
to
neutrophil
elastase
α1-­‐AT
inhibits
elastase
thus
preven.ng
loss
of
lung
elas.city
•
individuals
who
are
homozygotes
for
mutant
α1-­‐AT
are
very
suscep.ble
to
emphysema

88. Enzymes

91. Enzymes are
biological catalysts.

92. Some nomenclature…
Active site = special pocket where substrate binds
Specificity
1. enzymes are specific for a single molecule or a
structurally related group of substrates
2. usually only 1 enzyme per reaction type

93. Some more nomenclature…
Cofactor = inorganic component needed for enzyme
function

94. Some more nomenclature…
Coenzyme = nonprotein small organic component
needed for enzyme function

95. Some more nomenclature…
Holoenzyme - the enzyme protein plus its cofactor
Apoenzyme - enzyme protein without its cofactor
Prosthetic groups – a coenzyme that’s very tightly
(usually covalently) attached to
the protein, such as heme

99. How Enzymes Work
Enzymes increase the rate of reactions without
themselves being altered in the process of
substrate conversion to product.
This defines a catalyst.
Enzymes increase reaction rates by lowering the
energy input needed to form a reactant complex
that will eventually form product.
This occurs via the formation of a complex
between enzyme and substrate (ES):
k1 k2
E + S ES E + P
k-1

100. Steps in an Enzymatic Reaction
1. Enzyme and substrate combine to form a
complex.
2. Complex goes through a transition state – not
quite substrate or product
3. A complex of the enzyme and the product is
produced.
4. Finally, the enzyme and product separate.
All of these steps are equilibria.

101. Steps in an Enzymatic Reaction

102. Steps in an Enzymatic Reaction
1. Enzyme and substrate combine to form a
complex.

103. Steps in an Enzymatic Reaction
2. The complex goes through a transition state –
not quite substrate or product

104. Steps in an Enzymatic Reaction
3. A complex of enzyme and product is produced
(EP).
4. The product is released.

105. Factors that influence enzyme activity
Environmental factors
• temperature, pH
Cofactors
• metal ions
Effectors
• species that alter enzyme activity

106. Effect of pH on enzyme activity

107. Effect of pH on enzyme activity
Examples of optimum pH

108. Effect of temperature on enzyme activity
• exceeding normal temperature ranges always
reduces enzyme reaction rates
• optimum temperature is usually 25 - 40 ºC (but
not always)

109. Kinetics
• Kinetics is the study of the rate of change of
reactants to products
• Velocity (v) refers to the change in conc. of
substrate or product per unit time
• Rate (k) refers to the change in total quantity (of
reactant or product) per unit time
• Initial velocity (v0) is the change in reactant or
product conc. during the linear phase of a reaction

110. Michaelis-Menten Kinetics
Three basic assumptions:
1: ES complex is in a steady state, i.e.
remains constant during the initial phase of a
reaction
2: when enzyme is saturated all enzyme is in the
form of ES complex
3: if all enzyme in ES then rate of product
formation is maximal:
Vmax = k2[ES]

112. Michaelis-Menten Kinetics
The Michaelis-Menten equation is a quantitative
description of the relationship between the rate of
an enzyme catalyzed reaction (v1), substrate
concentration [S], the M-M rate constant (Km) and
maximal velocity (Vmax)

113. Michaelis-Menten Kinetics
Km is equal to the concentration of substrate
required to attain half maximal velocity for any
given reaction

116. Lineweaver-Burk Analysis
• Lineweaver and Burk manipulated the MM
equation by taking its reciprocal values generating a
double reciprocal plot
• Leads to a linear graph of the reciprocals of
velocity and substrate concentration

117. Lineweaver-Burk Plot

118. Enzyme inhibition
• many substances can inhibit enzyme activity:
substrate analogs
toxins
drugs
metal complexes

119. Enzyme inhibition - 2 broad classes:
Irreversible inhibition
• forms covalent or very strong noncovalent bonds
• site of attack is amino acid group that participates
in the normal enzymatic reaction
Reversible inhibition
• forms weak, noncovalent bonds that readily
dissociate from an enzyme
• the enzyme is only inactive when the inhibitor is
present

120. Enzyme inhibition
Competitive inhibitor
• resembles the normal substrate and competes
for the same site

121. Enzyme inhibition
Examples of competitive inhibitors:
• methanol and ethylene glycol compete with
ethanol for the binding sites to alcohol
dehydrogenase
• methotrexate competes with folic acid for
dihydrofolate reductase

122. Enzyme inhibition
Noncompetitive inhibitor
• materials that bind at a location other than the
normal site
• results in a change in how the enzyme performs

123. Enzyme inhibition
Examples of noncompetitive inhibitors:
• physostigmine is a cholinesterase inhibitor used
in the treatment of glaucoma
• captopril is an ACE inhibitor used in treatment of
hypertension
• allopurinol is a xanthine oxidase inhibitor used to
treat gout

124. Enzyme inhibition
Irreversible inhibitors
• permanently inactivate enzymes
• heavy metals (Hg2+, Pb2+, Cd2+)
• aspirin acetylates
• fluorouracil
• organophosphates

125. Enzyme Inhibition - Summary
Competitive
• Inhibitor binds at substrate site, inhibition is reversible as higher substrate
competes for inhibitor, Vmax unchanged, Km increased
Noncompetitive
• Inhibitor binds at site other than substrate, ESI cannot form product, increased
substrate does not compete, Km unchanged, Vmax decreased

126. Competitive Inhibition

128. Uncompetitive Inhibition

129. Noncompetetive model

130. Enzyme Regulation
• Proteolytic cleavage to activate:
Enzyme exists in inactive form (zymogen) that is
activated by removal of a short peptide segment ( truncation)
• Covalent modification to increase or decrease
activity, most common is phosphorylation
• Sequestration: enzyme forms inactive polymers
• Allosteric (“other site”) regulation, both positive
and negative ( homotropic, heterotropic)
Induction-upregulation: increase gene expression, synthesis of more enzyme
molecules
Repression-downregulation: decrease gene expression, decrease synthesis of
enzyme molecules.

131. Allosteric enzymes
Are regulated by molecules called effectors
(modifiers) that bind non-covalently at a site
other than active site. They can alter Vmax or
Km or both)
1. Homotrophic effectors – when the substrate
itself is an effector
2. Heterotrophic effector – when the effector is
different from a substrate (often it is an end-
product - feedback inhibition)

132. Allosteric enzymes show sigmoid curve
(cooperative substrate binding like in Hb)

134. Feedback inhibition

137. Enzymes Used in Clinical diagnoses
Tissue damage: Increased release of tissue enzymes in plasma
Enzyme assay is used for both diagnostic and prognostic purpose
Eg: ALT – present in the liver will be appearing in the plasma if there is
Liver damage or cell necrosis
Isoenzymes: Structurally different enzymes but catalyze the same reaction
Eg: CK1, CK2, CK3 (creatine kinase, CK MB (CK 2) is present in the heart, its
presence in plasma is indicative of myocardial infarction

138. ALSO: Troponin T & Troponin I
are also released in cardiac
damage. Peaks in 8 – 24hr
Sensitive and specific for cardiac
tissue damage