Download Factors that Affect Enzyme Catalysis

Factors that Affect Enzyme Catalysis
Ionic strength: µ = ½ Σ (Zi2ci)
Review: urea, guanidinium
hydrochloride, detergents, or
organic solvents
Temperature
pH
Factors that Affect Enzyme Catalysis: µ
Ionic strength: µ = ½ Σ (Z i2 ci)
Cl- competes
with O2 - for
active Cu2+
Superoxide Dismutase
k (107 M-1 s-1 )
10.0
Cu+2 + O2 -
à
Cu+ + O2
Cu+ + O2 - + 2 H+ à Cu+2 + H2 O2
1.0
0.0
2.5
NaCl (mM)
5.0
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Conditions that cause protein denaturation:
Urea/Guandinium.HCl
C O 2-
O
C
H2N
R
R
NH2+ ClNH2
H2N
R
R
C
NH2
denaturation
C O 2-
R
renaturation
N H 3+
N H 3+
Folded protein
R
Unfolded protein
-Addition of urea or guanidinium hydrochloride (G@@HCl)
-Urea and G@@HCl both H-bond to proteins causing
disruption of the H-bond stabilization.
-Proteins unfold and remain soluble (usually).
Conditions that cause protein denaturation:
Soaps/Detergents
O
C O 2-
R
CH3 CH2CH2CH2 CH2CH2CH2 CH2 CH2 CH2 CH2 C
denaturation
R
R
R
C
R
renaturation
N H 3+
Folded protein
R
N H 3+
O
Na
O 2-
R
R
Unfolded protein
R
NH 3
+
C O 2-
rod-like structures formed in
sodium dodecylsulfate (SDS)
Addition of soaps or detergents
Nonpolar portions of soaps and detergents
interact with protein R groups causing the
loss of the hydrophobic stabilization.
Proteins denature and remain soluble.
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Conditions that cause protein denaturation:
Organic Solvents
C O 2-
R
R
denaturation
R
R
R
see below
C O 2-
R
R
N H 3+
R
C O 2-
renaturation
R CO 2
R
R
N H 3+
N H 3+
Folded protein
N H 3+
R
protein precipitates
with poorly defined
structure
Unfolded protein
Addition of water-soluble organic solvents
CH3CH2 O
H
Solvent molecules interact with protein R
groups causing the loss of the
hydrophobic stabilization.
O
CH3
C
CH3
Proteins denature and often precipitate.
Conditions that cause protein denaturation: H+/OHC O 2-
R
denaturation
R
R
R
se e below
C O 2-
R
R
R
N H 3+
R
C O 2-
renaturation
R
N H 3+
N H 3+
Folded protein
R CO 2
R
N H 3+
R
Unfolded protein
prot ein prec ipitates
wit h poorly defined
st ructure
H
O
H
O
H
H
Extremes of pH
-OH - and H 3O+ both H-bond to proteins causing disruption
of the H-bond stabilization.
-Some proteins may unfold and aggregate into
denatured precipitates (solids). This is more common at
low pHs.
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Factors that Affect Enzyme Catalysis: Temperature
Temperature
http://www.bio.mtu.edu/campbell/bl482/lectures/lec2/482enz2.htm
Factors that Affect Enzyme Catalysis: pH
(NOT EXTREMES OF pH)
Bell-shaped pH-rate profiles
Fig 8-17a,b
The enzyme active site has a minimum of two functional groups.
For the maximum rate:
One group is required in the conjugate acid form, AH
One group is required in the conjugate base state, B:
rate = k[E-AH][E-B:]
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rate = k[E-AH][E-B:]
Fig 8-17a,b
Examples of AH = -HISH+, -CO2 H, -SH, -NH3 +, etc
Examples of B: = -HIS:, -CO2 -, -S-, -NH2 , etc
E-AH
+H:B-
inactive
E-AH
º :B-
active
E-A-
º :B-
inactive
Active form has a maximum concentration
at the top of the bell-shaped profile
What amino acids are likely to be involved in catalysis?
Fig 8-17a,b
E-AH
+H:B-
inactive
E-AH
º :B-
active
E-A-
º :B-
inactive
The pKa values for the functional groups
involved in catalysis can be estimated by
looking at the pH values on either side of the
bell profile at ½ maximum velocity
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PEPSIN?
Fig 8-17
pKa values indicate
very acidic groups
-ASP -- CO2H
-GLU -- CO2H
E-AH
+H:B-
E-AH
º :B-
inactive
active
E-A-
º :B-
inactive
GLUCOSE-6-PHOSPHATASE?
Fig 8-17
pKa values indicate what amino acids?
pKa ~ 9.5
LYS or N-terminal (what form?)
CA since rate 9 with pH 8
pKa ~ 6
HIS (what form?)
CB since rate 8 with pH 8
E-AH
+H:B-
inactive
E-AH
º :B-
active
E-A-
º :B-
inactive
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Chymotrypsin
Chymotrypsin
%
Rate
%
Rate
?
E-B:
What amino acid side
chain and what form of
this side chain (CA or CB)
accounts for this increase
in rate as pH is raised?
histidine
his
H
CH2
rate % [E-HIS:]
HN
NH
HN
pKa = 6.0
E-HIS:H +
inactive
CH2
N
+ H+
E-HIS:
active
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Chymotrypsin
Why does rate level off at high pH?
histidine
his
H
CH2
HN
NH
HN
pKa = 6.0
E-HIS:H +
CH2
N
+ H+
E-HIS:
inactive
active
rate % [E-HIS:]
[Eo] <<<< [So] at all So; Eo is the limiting reagent.
When all of the E-HIS is in the CB form, the maximum
amount of the CB form is present.
Since the rate [E-HIS], the rate reaches a maximum value.
Problem Set: Notation
CHEMICAL BASIS OF ENZYME CATALYSIS
In the attached schemes, the enzyme active site is schematically represented with a bold-lined "box".
The amino acid side chains that act as functional groups i the site are written in bold lines and letters
and are attached to the "box". Substrates are indicated in plain text.
Example substrate:
O
Example enzyme "box"
for chymotrypsin:
C
O
NOTATION:
H
N
N
O
R'
NH
H
C
O
R
CH 2
hydrogen bonding or other
weak interactions
mechanistic arrows
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Problem Set: Catalytic Triad
Catalytic Triad of
Serine Proteases
ASP- ... HIS: ... SER
Problem Set: Sample Step #1
CHYMOTRYPSIN
O
R'
NH
O
ASP
CB
C
O
H
N
H
N
C
R'
O
C
O
H
N
R
O
HIS
CB
Peptide
Substrate
CH2
O
C
NH
SER
Nu
Covalent
R
Intermediate
O
N
H
CH2
H O
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Problem Set: Sample Step #2
R'
O
ASP
CB
C
O
H
N
NH
H
O
R'
O
O
C
N
Intermediate
O
N
SER
NH2
R
H
H
R
CH2
H2 O
H
O
Covalent
C
O
N
HIS
CA
C
O
CH2
Problem Set: What is function of ASP?
CHYMOTRYPSIN
1. Keeps HIS oriented.
HIS:H+
2. Raises
pKa making
+
CA-HIS:H a poorer acid,
but the CB-HIS: a better
base to attach the very weak
acid HO-SER!
O
R'
NH
O
C
O
H
N
H
N
R'
O
C
O
H
N
C
R
O
CH2
O
C
NH
R
O
N
H
CH2
HO
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Problem Set: Predict pH-Rate Profile
CHYMOTRYPSIN
O
R'
C
O
H
N
C
NH
O
H
N
R
O
CH2
O
R'
O
C
O
H
C
NH
R
O
N
N
H
CH2
HO
Deacylation of Chymotrypsin: pH-Rate Profile
H
O
C
O
C
R
H
O
H
N
O
N
H
O
C
2
O
O
H
N
CH 2
O
C
O
R
O
N
H
CH 2
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More about Chymotrypsin
CHYMOTRYPSIN CATALYSIS OF
PEPTIDE AND ESTER HYDROLYSIS
PING PONG BI BI MECHANISM
P1
S1
S2
E'
E
P2
E
Ping Pong Mechanism!
O
O
R C NR'
H
S1
H NR'
H
H2O
S2
P1
E OH
R C OH
P2
E' O C R
ser
E OH
O
fast
acyl-enzyme intermediate
Fast = acylation step
slow
Slow = deacylation step
Acyl enzyme = inactive
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Understanding Laboratory Experiment
slow
P2
S2
E
S1
E'
P1
fast
slow formation of P 1
[P1]
[P1]
t = o = [Eo]
= active sites
fast formation of P 1 0.0
ms
3.0
time (min)
Read Text about Chymotrypsin
Fig 8-20
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Read Text about Chymotrypsin
etc.
Structure-Activity Relationships
Box 8 -3
B compared to A: B faster, tigher binding
C compared to B: C faster, weaker binding
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Enzyme Activity
Vo (rate) at given Eo has units of M min -1
Specific Activity = Rate/mg Eo
M min -1
-------- =
mg
U
---mg
1 U often defined as 1 m mol/min
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