Download Effect of Temperature Increasing the temperature increases the

Effect of Temperature
Increasing the temperature increases the
energy in the system
Two effects
• kinetic
• denaturing
Kinetic effect
• Increased motion of molecules
• Increased collisions between enzyme/substrate
• Increased rate of reaction
2x increase in rate
for every 10 ºC
rise in temperature
Rate
Temperature
Denaturing effect
• Proteins take on the 3-D structure with
lowest potential energy - increases their
stability
• Increased energy causes increased motion
within the molecule as well as between
molecules
• Weak bonds in the tertiary structure
(hydrogen bonds) are broken and new
bonds form in different positions
• New 3-D structures form
If the change in 3-D structure alters the
active site of the enzyme so that it cannot
catalyse the reaction it is said to be denatured
100
% not
denatured
0
Temperature
Overall effect of temperature
Kinetic
effect
dominant
Optimum
Activity
Temperature
Denaturing
effect
dominant
Most enzymes have an Optimum Temperature
at which they work best
Optimum temperature is not always ~37 ºC
Denaturing effect is due to both temperature
and time of exposure to that temperature
Effect of varying substrate concentration
• At low substrate concentrations [S]
• If the number of enzyme molecules [E]
remains constant
• The number of substrate molecules
present [S] determines how fast the
reaction takes place
[Rate of reaction = activity = IRV = v]
V  [S] (First
order kinetics)
v
[S]
• At high substrate concentrations [S]
• If the number of enzyme molecules [E]
remains constant
• The number of enzyme molecules
present [E] determines how fast the
reaction takes place
[Rate of reaction = activity = IRV = v]
V  [S] (Zero
order kinetics)
v
[S]

V  [S]. Zero
order kinetics
V  [S]. First
order kinetics
v
Hyperbolic curve typical of simple
enzymes
(Michaelis-Menten
kinetics)
[S]

Michaelis-Menten
v=
Vmax[S]
Km + [S]
v = IRV at a specified [S]
Vmax = maximum IRV attainable by the
enzyme under given conditions
[S] = substrate concentration
Km = Michaelis constant
v
Vmax
Km - [S] at half Vmax
Indicator of affinity of
enzyme for its substrate
High Km - low affinity
Low Km - high affinity
Vmax/2
Km
[S]

v
Vmax
Enzyme 1
Enzyme 2
Vmax/2
Km
Km
[S]
• Both enzymes give same Vmax
• Enzyme 1 needs lower [S] to reach Vmax/2 so
has higher affinity for substrate
• Enzyme 1 has lower Km
• Plateau on Michaelis-Menten graph only
truly reached at infinitely high [S]
• Cannot carry out experiments at those high
concentrations in lab
• Cannot determine Vmax and Km
experimentally by plotting v against [S]
• Use a derivation of Michaelis-Menten
• It is the inverse of the Michaelis-Menten
equation
• Called the Lineweaver-Burk equation
Lineweaver-Burk
1/v
1/Vmax
Gradient = Km/Vmax
1/[S]
-1/Km
1 = Km
v
Vmax[S]
+
1
Vmax
Where is the register?
ENZYMES
• Proteins (biological macromolecules)
• Catalysts
• Show specificity
• Sensitive to changes in physical and
chemical environment
• Their activity can be controlled
Enzyme Inhibitors
Reduce the rate of an enzyme catalysed
reaction
• Irreversible
• Reversible
• Competitive
• Non-competitive
• Uncompetitive
Irreversible inhibitors
• Bind irreversibly to enzyme
• Usually bind via a covalent bond
• Bind to an amino acid side chain at or
near the active site
• Commonly bind to either Ser (-CH2-OH)
or Cys (-CH2-SH) side chains
• Binding permanently inactivates the
enzyme
• Usually prevents substrate binding
DFP (di-isopropylfluorophosphate)
• Nerve poison.
• Covalently binds
to a Ser residue
in acetylcholine
esterase
• Prevents
breakdown of the
neurotransmitter
acetylcholine
Ser
OH
F
H3 C
CH3
CH O P O CH
CH3
H3 C
O
+HF
Ser
O
H3C
CH3
CH O P O CH
CH3
H3C
O
F
DFP
H3C
CH O
P
CH3
O
O
CH
CH3
CH3
F
Sarin
H3C
CH O
P
CH3
O
CH3
Insecticides – eg. Malathion, parathion
S
H 3C
O
P
O
CH3
O
S
CH
C
O
C 2H 5
CH2 C
O
C 2H 5
O
Penicillin • Antibiotic
• Covalently binds to a Ser residue in
glycopeptide transpeptidase
• Prevents synthesis of bacterial
cell wall peptidoglycan
R
C O
NH
C N
O
OH
Ser
S
CH3
CH3
R
C O
NH
COOH
O C N
H
Ser
S
CH3
CH3
COOH
Polysaccharide
Tetrapeptide
Penta-Gly bridge
Competitive Inhibitors
• Reversible inhibitor
• Compete with substrate for access to
active site
• Often have structure similar to substrate
• When bound to enzyme prevents binding
of substrate
• Can be overcome by increasing [S] until it
out-competes inhibitor
v
1/v
Vmax
Vmax/2
1/Vmax
Km Km
E+S
+
I
EI
[S]
ES
-1/Km
-1/Km
1/[S]
E+P
Km increases
Vmax remains unchanged
COOH
CH2
COOH
Malonate
H2N
COOH
CH2
CH2
COOH
COOH
CH
CH
COOH
Succinate
Fumarate
NH 2
C O
para aminobenzoic acid
H2N
NH 2
S O
Sulphanilamide
Non-Competitive Inhibitors
• Reversible inhibitor
• Bind at a site other than the active site
• Bind before or after the substrate binds
• Do not prevent the substrate from binding
• Prevent catalytic act from taking place
• Cannot be overcome by increasing [S]
• Can be removed by repeated dialysis
Reaction of the -SH group of Cys with a
heavy metal ion eg. Hg2+, Pb2+, Ag+
v
Vmax
1/v
Vmax/2
Vmax
Vmax/2
1/Vmax
1/Vmax
Km
[S]
E+S
+
I
ES
+
I
EI+ S
EIS
-1/Km
1/[S]
E+P
Km remains unchanged
Vmax decreases
Next week
Wednesday 11 – 12
Surgery 2 – Enzymes
Thursday 5-6
Assessment practice 1 Proteins and enzymes
You will get 25 min to write an answer to the sort of question you
might get in the exam in January. There will then be 25 min in which
I will go over what the answer should be so you will get feedback on
how you performed
To help you prepare there are two self-assessment “tests” available:
a paper-based “test” and an on-line “test” which you can do as
often as you want.
Available in the Lecture Support folder on Blackboard