L34 2009 Enzymes

L34 2009 Enzymes - David S Goodsell of The Scripps Research...

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Unformatted text preview: 11/16/09 David S. Goodsell of The Scripps Research Institute En m zy es Enzymes
 ‐ 
Regulate
cellular
functions
by
determining
 the
velocity
(speed)
of
cellular
reactions
 ‐ 
Large,
globular
proteins
folded
into
complex
 3D
shapes
(conformations)
 Substrate
 David S. Goodsell of The Scripps Research Institute Active
site
 NA SA NASA Opabinia regalis Outline
 •  Enzymes
and
How
they
Work
 •  Enzyme
Kinetics
 •  Spectrophotometry
 •  Use
of
a
Standard
Curve
 •  Competitive
and
Noncompetitive
Inhibition
 Free
energy
(G)
changes
in
an
exergonic
reaction
 AB
+
CD

AC
+
BD

 Enzymes reduce activation energy Substrates
maintained
in
correct
 orientation
 H2O

 Reaction
rate
 H+

+

OH‐
 Enzyme
1
 pH
=
‐log10[H+]
 Enzyme
2
 Substrate
electron
distribution
 altered,
stabilizing
transitional
 state

 pH
 Causing
physical
stress
on
 chemical
bond
to
be
broken
 In
non‐ideal
pH,
enzyme
conformation
changes.
 An
enzyme
loses
its
ability
to
bind
its
substrate
 and
lower
activation
energy.
 1 11/16/09 Enzyme
Kinetics
 Leonor
Michaelis
 (1875‐1949)
 1 Substrates enter active site; enzyme changes shape such that its active site enfolds the substrates (induced fit). 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. Substrates Enzyme-substrate complex 3 Active site can lower EA and speed up a reaction. Maud
Menten
 (1879‐1960)
 E
+
S








ES








E
+
P
 The
study
of
the
rate/velocity
at
which
an
enzyme

 Works
and
the
variables
that
affect
this
rate
 6 Active site is available for two new substrate molecules. Enzyme E
+
S








ES
(






EP)








E
+
P
 5 Products are released. 4 Substrates are converted to products. Products Kinetics
:
Law
of
Mass
Action
vs.
Enzyme‐Catalysis
Models 
 Velocity
of
reaction
 (μM/min
of
product)

 Velocity
of
reaction
 (μM/min.
of
product)

 Vm
 Reaction
rate
with
an
enzyme
 
 The
maximum
velocity
(Vmax
or
Vm)
 
Vm
 2
 Z Substrate
concentration
(mM)
 Km
 The
Michaelis‐Menten
Constant
(Km)
 equals
the
substrate
concentration
 required
to
obtain
half
Vm

 Substrate
concentration
(mM)
 Velocity
of
reaction
(μM/min
of
product)
 Velocity
of
reaction
(μM/min
of
product)
 Reaction
rates
of
different
enzymes
 Vm
 Different
Concentrations
of
Same
Enzyme 
 Vm
with
[
E
]=
1.0
mg /ml

 Vm
 2
 .
 
Vm
 with
 2
 [
E
]
=
1.0
 
Vm
 with
 2
 [
E
]
=
0.5
 Vm
with
[
E
]=
0.5
mg /ml

 Different enzymes have different Kms 
B
 K
A

 Km
 Substrate
concentration
(mM)
 m Different concentrations of an enzyme have different Vm but same Km Km
 Substrate
concentration
(mM)
 2 11/16/09 TECHNIQUE
 Spectrophotometry
 The
measurement
of
the
proportions
of
 light
of
different
wavelengths
transmitted
 or
reflected
(or
absorbed)
by
a
pigment.
 
…
allows
one
to
estimate
the
amount
of
a
 substance
in
solution
by
measuring
the
 intensity
of
light
transmitted
by
the
 solution.
 White
 light
 Refracting
 prism
 Chlorophyll
 Photoelectric
 solution
 tube
 2
 3
 4
 Galvanometer
 1
 Slit
moves
to
 pass
light
 of
selected
 wavelength
 Green
 light
 The
high
transmittance
 (low
absorption)
 reading
indicates
that
 chlorophyll
absorbs
 very
little
green
light.
 Blue
 light
 The
low
transmittance
 (high
absorption)
 reading
indicates
that
 chlorophyll
absorbs
 most
blue
light.
 Absorption
spectrum
of
p‐nitrophenol
 1.0
 Spectrophotometry
 A = -(log10T) Absorbance
 0.5
 Spectronic
20TM
 0.0
 400
 500
 600
 700
 Wavelength
of
visible
light
(nm)
 Spectrophotometry
 e.g.
for
10%
T,

A=‐(log100.1)
=
1
 
























…
since
10‐1
=
0.1
 Alkaline
phosphatase
 Spectronic
20TM
 Transmittance
decreases
exponentially
with
 the
concentration
of
a
light‐absorbing
 substance,
whereas
absorbance
varies
 directly
with
concentration
and
A
=
‐logT
 3 11/16/09 Alkaline
phosphatase
 • 
Removes
phosphate
group
from
 5´
end
of
nucleotides
and
proteins

 • 
Functions
in
basic
environment
 but
denatured
at
pH
=
12
 • 

Use
Glycine/NaOH
buffer
‐
pH
 In
our
experiment…
 ‐ 
Select
a
special
substrate,
that
gives
us
a
colored
product
 Substrate
 Enzyme
 Product
 range
of
8.6
to
10.6
(ours
=
10.5)
 Alkaline
 
 phosphatase 
 P‐nitrophenyl
phosphate 
 
 
 
 

 



p‐nitrophenol





phosphate
 Standard curves are used to determine the value of an unknown quantity from a more easily measured characteristic 1) begin with something you know - e.g. concentration of a substance (standards) 2) relate #1 to a measurable physical characteristic - e.g. transmittance, absorbance 3) Graph the observed relationship between know concentrations and measured absorbance to make a standard curve 4) Use the standard curve to estimate the concentration of an unknown using the measurable absorbance Abs Procedure
highlight
 ‐ 
Each
group
works
on
one
specific
[
E
]
 ‐ 
A
series
of
7
[
S
],
each
paired
with
a
blank
 ‐ 
the
blank
has
everything
except
the
product
formed
 ‐ 
compare
test
cuvette
with
blank
cuvette
 ‐ 
Allow
reaction
in
37˚C
water
bath
for
15
min.
 ‐ 
Stop
reaction
using
NaOH
(.2N,
pH
12.8)
 12 34 56 78 9 10 11 12 13 14 Procedure
highlight
 1)  Use
your
standard
curve
to
estimate
p‐nitrophenol
 concentration
from
calculated
absorbance
(from
 transmittance
data)
 2)

Convert
concentration
to
velocity
(divide
by
15
min)
 1
 blank blank blank blank blank blank blank Absorbance
 0
 Same
[
E
]
 Increasing
[
S
]
 [
p‐nitrophenol
]
 µM
 www.wellesley.edu/Biology/Concepts/Html/standardcurve.html 4 11/16/09 Fig. 8-19 Substrate Active site Competitive inhibitor Enzyme In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same reaction velocities that were reached in the inhibitor’s absence. This will change: A.  Vmax B.  Km C.  Vmax and Km D.  Neither Vmax nor Km E.  This is too confusing without
inhibitor
 Noncompetitive inhibitor (a) Normal binding (b) Competitive inhibition (c) Noncompetitive inhibition Competitive
Inhibition
 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
 Velocity
of
reaction
 (μM/min
of
product)

 without
inhibitor
 Noncompetitive
Inhibition
 ‐ 
Inhibitor
binds
to
enzyme
away
from
its
active
site
 causing
conformational
change
 ‐ 
As
if
total
amount
of
functional
enzymes
reduced
 Vm
 Velocity
of
reaction
 (μM/min
of
product)
 Vm
 With
competitive
 inhibitor
 without
inhibitor
 Vm
I
 
 with
noncompetitive
 inhibitor
 Vm
 2
 Km
 Km
 
I
 Substrate
concentration
(mM)
 Km
 Substrate
concentration
(mM)
 David S. Goodsell of The Scripps Research Institute David S. Goodsell of The Scripps Research Institute Opabinia regalis NA SA NASA 5 ...
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