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Stickase
Substrate
Transition state
X
Product
If enzyme just binds substrate
then there will be no further reaction
Enzyme not only recognizes substrate,
but also induces the formation of transition state
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
Factors affecting Enzymes
•
substrate concentration
In non-enzymic reactions the increase in velocity is proportional
to the substrate concentration.
Enzymic reaction is faster but it reaches a saturation point when all
the enzyme molecules are occupied. If you alter the concentration
of the enzyme then Vmax will change too.
• pH
Extreme pH levels will produce denaturation and the substrate
molecules will no longer fit in active site.
Small changes in ionisation and in the charges of the enzyme and
it’s substrate molecules will affect the binding of the substrate with
the active site.
•
temperature
The optimum temperature for an enzyme controlled reaction will be
a balance between the Q10 and denaturation. (Q10 - temperature
coefficient - increase in reaction rate with a 10°C rise in temperature.
•
inhibitors
Enzýmová reakcia pri rôznych koncentráciách substrátu
EXTREMOPHILES
For most enzymes the optimum
temperature is about 30°C, many are
a lot lower, e.g. cold water fish will die
at 30°C because their enzymes
denature.
A few bacteria have enzymes that can
withstand very high temp. up to 100°C.
Most enzymes however are fully
denatured at 70°C.
t
vo = [P] / min
Unit = mmole/min
Slope
tan
0
10
20 30
40
Reaction time (min)
Specific Activity Units
Activity = Protein (mg)
y
x
y
= tan
x
Juang RH (2004) BCbasics
S → P mmole
Product [P]
Enzyme Activity Unit
Significance of Enzyme Kinetics
v0 = Vmax × K
Obtain
Vmax and Km
1st order
[S] = Low → High
Vmax [S]
Km + [S]
E3
E2
E1
Proportional to
enzyme concentration
zero order
vo =
= k3 [Et] × K
[S] = Fixed concentration
Juang RH (2004) BCbasics
1
2
3
4
5
6
7
8
S
+
E
↓
P
80
60
40
20
0
0
2
4
6
8
Substrate (mmole)
(in a fixed period of time)
Product
Increase Substrate Concentration
0
Juang RH (2004) BCbasics
An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme
→E
2) Add substrate in various concentrations → S (x)
3) Measure Product in fixed Time (P/t)→ vo (y)
4) (x, y) plot get hyperbolic curve, estimate → Vmax
5) When y = 1/2 Vmax calculate x ([S])
→ Km
Vmax
1
vo
1/2
-1
Km
1
Vmax
Double reciprocal 1/S
Km Direct plot S
Juang RH (2004) BCbasics
vo
A Real Example for Enzyme Kinetics
Data
Substrate Product
Velocity
Double reciprocal
[S] Absorbance v (mmole/min)
0.25
0.21 →
0.42
0.50
0.36 → 0.72
1.0
0.40 → 0.80
2.0
0.46 →
0.92
no
1
2
3
4
1/S
4
2
1
0.5
1/v
2.08
1.56
1.35
1.16
1.0
v
0.5
0
0
1
2
[S]
2.0
1.0
1/v
1.0
-3.8
0
-4
-2
0
2
1/[S]
4
Juang RH (2004) BCbasics
Double reciprocal
Direct plot
(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole
(2) Reaction time was 10 min
Enzyme Inhibitors
- chemicals that reduce the rate of enzymic reactions
- usually specific, they work at low concentrations
- block the enzyme but they do not usually destroy it
Irreversible inhibitors: Combine irreversibly with the
functional groups of the amino acids in the active site, e.g.
nerve gases and pesticides, containing organophosphorus, combine with serine
residues in the acetylcholine esterase.
Reversible inhibitors: These can be washed out of the
solution of enzyme by dialysis.
- competitive inhibitor competes with the substrate molecules for the active site.
The inhibitor’s action is proportional to its concentration. Resembles the substrate’s
structure closely.
- non-competitive inhibitor is not influenced by the concentration of the substrate. It
inhibits by binding irreversibly to the enzyme but not at the active site, e.g. cyanide
combines with the Iron in the enzymes cytochrome oxidase, heavy metals Ag or Hg
combine with –SH groups. These can be removed by using a chelating agent such
as EDTA.
Negative feedback: end point or end product inhibition
Poisons snake bite, plant alkaloids and nerve gases
Medicine antibiotics, sulphonamides, sedatives, stimulants
Affinity labeling of enzymes
Enzyme Inhibition (Mechanism)
Equation and Description
Cartoon Guide
I
Competitive
I
Non-competitive
Substrate
E
S
S
E
I
Compete for
Inhibitor active site
S
I
I
Uncompetitive
S
E
I
I
Different site
E + S←
→ ES → E + P
+
I
↓↑
EI
E + S←
→ ES → E + P
+
+
I
I
↓↑
↓↑
EI + S →EIS
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
S
I
E + S←
→ ES → E + P
+
I
↓↑
EIS
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
Juang RH (2004) BCbasics
Enzyme Inhibition (Plots)
I
Competitive
I
Non-competitive
Direct Plots
Vmax
vo
vo
I
Double Reciprocal
Km Km’
I
[S], mM
Km = Km’
I
Uncompetitive
Vmax
Vmax
Vmax’
Vmax’
[S], mM
I
Km’ Km
[S], mM
Vmax unchanged
Km increased
Vmax decreased
Km unchanged
Both Vmax & Km decreased
1/vo
1/vo
1/vo
Intersect
at Y axis
1/Km
I
I
I
Two parallel
lines
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
1/ Vmax
1/[S]
1/ Vmax
1/Km
1/[S]
Juang RH (2004) BCbasics
Enzyme Kinetics
kcat /Km
vo= Vmax [S]
Km + [S]
kcat
Significance
zero order
1st order
Observe vo change
under various [S],
resulted plots
yield Vmax and Km
Turn over
number
Vmax
k3 [Et]
1 mmole
min
Specific Activity
Activity
&
E3
E2
E1
Km
Affinity with
substrate
Double reciprocal
Bi-substrate reaction also
follows M-M equation, but
one of the substrate should
be saturated when estimate
the other
Inhibition
Maximum
velocity
Activity Unit
unit
mg
Direct plot
Competitive
Non-competitive
Uncompetitive
Juang RH (2004) BCbasics
Enzýmová analýza
Niektoré enzýmy
dôležité pre klinickú
diagnostiku