Option B Enzyme Kinetics, Pigment and Anthrocyanin electron conjugation

1. Evaluate specificityof enzyme for substrate
Low Km – High affinity
High Km – Low affinity
SK
SV
v
m 

][max
Enzyme kinetics
Michaelis Menten eqn
Enzyme Enzyme/substratecomplex Product
Rate
Sub conc
Max rate velocity
2
maxV
Km 
Km = [S] when rate
is half Vmax
Rate
Sub conc
High [S] conc – enzyme saturated
– all active sites not available
(zero order)
Low [S] conc - rate proportional to [S]
- all active sites available
(1st order)
Saturation occurs in formation of complex
Rate
Sub conc
Enzyme A
Enzyme B
Km – Low
Rate HIGHER
High affinity
at low [S] conc
Km - High
Rate LOWER
Low affinity
Km
Km
mK
SK
SV
v
m 

][max
Low [S]
Km >[S]
High [S]
Km <[S]
][
][
max
max
S
K
V
v
K
SV
v
m
m


(1st order) rate prop to [S]
All active site available
max
max
max
][
][
Vv
S
S
V
v
SK
SV
v
m




(zero order) to [S]
All active site saturated
constant

2. Evaluate specificityof enzyme for substrate
Low Km – High affinity
High Km – Low affinity
SK
SV
v
m 

][max
Enzyme kinetics
Michaelis Menten eqn
Enzyme Enzyme/substratecomplex Product
Rate
Sub conc
Max rate velocity
2
maxV
Km 
Km = [S] when rate
is half Vmax
Rate
Sub conc
High [S] conc – enzyme saturated
– all active sites not available
(zero order)
Low [S] conc - rate proportional to [S]
- all active sites available
(1st order)
Saturation occurs in formation of complex
Rate
Sub conc
Enzyme A
Enzyme B
Km – Low
Rate HIGHER
High affinity
at low [S] conc
Km - High
Rate LOWER
Low affinity
Km
Km
mK
m
cat
K
K
efficiencycatalytic .
 mcat KK
Kcat = turnover number- max sub convert to product per second (Enzyme saturated)
Click here view KM Click here Michaelis Menten

3. CompetitiveInhibitor
Rate
Sub conc
Max rate velocity
Km = [S] when rate
is half Vmax
Rate
SK
SV
v
m 

][max
Low [S]
Km > [S]
High [S]
Km < [S]
][
][
max
max
S
K
V
v
K
SV
v
m
m


(1st order) rate prop to [S]
All active site available
max
max
max
][
][
Vv
S
S
V
v
SK
SV
v
m




(zero order) to [S]
All active site saturated
Compete same active site
Structurally similar
Enzyme kinetics
No Inhibitors
Sub conc Sub conc
Non Competitive Inhibitor
enzyme
substrate inhibitorsubstrate
enzyme
Competitive
inhibition
Non-competitive
inhibition
Active site Bind active site Bind allosteric site
Effect Vmax No change Decrease
Effect Km Increase No change
Bind diff site (allosteric site)
Structurally different
inhibitor substrate
enzyme
Km ↑ - Enzyme affinity ↓
V max - No change
High [S] to achieve Vmax
V max – Lower ↓ Changed
Enzyme unavailable
Km - No change
Enzyme affinity unchanged
allosteric
site

4. Sub Conc Rate, v
0.02 10.8
0.04 18.5
0.07 26.7
0.1 32.5
0.15 39.2
0.2 43.3
0.3 48.7
0.5 54.4
Enzyme activity measured against substrate conc. Find Vmax and Km
Vmax
Sub conc
Rate
= 60
Km = 0.1
0.1 0.2 0.3 0.4
Vmax – Lower ↓ - Enzyme unavailable
Don’t alter active site – no effect on Km
Alter conformationalchangeenzymeNOT substratebinding(affinity)
Km - Unchange- Enzyme affinity unchanged
Vmax – Unchanged –High [S} will reduced inhibition
Compete forsame active site - substrate binding (affinity)lower ↓
Km - Change- Enzyme affinitylower ↓
Vmax
Vmax
Km Low Km High
Sucrose conc Rate
No inhibitor
Rate
Inhibitor
0.029 0.181 0.095
0.058 0.266 0.140
0.088 0.311 0.165
0.117 0.338 0.180
0.175 0.369 0.197
Vmax
Vmax
0.4
0.2
Vmax – Lower ↓ - Enzyme unavailable
Don’t alter active site – no effect on Km
Km - Unchange- Enzaffinity unchanged
Km same
2
maxV
Km 
Rate
Sub conc

5. Sub Conc Rate, v
0.02 10.8
0.04 18.5
0.07 26.7
0.1 32.5
0.15 39.2
0.2 43.3
0.3 48.7
0.5 54.4
Enzyme activity measured against substrate conc
Find Vmax and Km
Vmax
Sub conc
Rate
= 60
Km = 0.1
0.1 0.2 0.3 0.4
At low sub conc, all active site free,
rxn directly proportional to sub conc
State /explain how rate enzyme
catalyzed rxn related to substrate conc
Compare enzymesandinorganic catalyst
Enzyme Catalyst
Similarity Both increase rate
Both lower activation energy
No effect on yield
Differences Protein Not protein
Show saturation kinetic
(hyperboliccurve)
Do not
(linear relationship)
Regulated by inhibitor Less likely
Sensitive to Temp/pressure Not affected
Rate
conc
Rate – 1st order
Rate zero order
At high sub conc, all active site saturated,
rate reach its max
2
maxV
Km 

6. C C
Absorption of UV by organic molecules and chromophores
Absorption UV radiation by
C = C, C = O, N = N, N =O gps
C = C /N = N (π bond)
C = O: (lone pair electron)
NO2 (lone pair electron)
Chromophores gps
Ground
Higher emptyorbital
π electron
Absorb UV to excite π/lone pair e to higheremptyorbital
C O
lone pair
electron
:
Chromophores – organic molecule with conjugated double bond
Absorb radiation to excite delocalized e to empty orbital
alternating double/single bond
Filled orbital Bonding orbital
empty orbital antibonding orbital
Biological Pigments (Anthocyanins)
Coloured – extensive conjugation of electrons
alternating single and double bond
Porphyrin Chlorophyll Heme (hemoglobin)
Anthocyanin
Carotene
absorb absorb absorb absorb

7. C C
Absorption UV radiation by
C = C, C = O, N = N, N =O gp
C = C /N = N (π bond)
C = O: (lone pair electron)
NO2 (lone pair electron)
Ground
π electron
Absorb UV to excite π/lone pair e to higheremptyorbital
C O
lone pair
electron
:
Absorb radiation to excite delocalized e to empty orbital
alternating double/single bond
Filled orbital Bonding orbital
empty orbital antibonding orbital
Carotene
Diff bet UV and Visible absorption
Colourless- Absorption in UV range
Electronic transitionfrom bondingto antibonding orbital
(involve pi / lonepair e)
UV visible
Organic molecules/chromophores
BiologicalPigments(Anthocyanins)
Coloured – extensiveconjugation of electron
Alternatingsingle and double bond
Electron in pi orbitaldelocalized through single and double bond.
π elec excitedby absorbinglong wavelength in visible region
Anthocyanin
Chlorophyll
absorb absorb absorb absorb
Higher emptyorbital

8. Absorb radiation to excite delocalized e to empty orbital
Filled orbital
empty orbital
Carotene
Colourless– Absorption in UV range
Electronic transition frombonding to antibonding orbital
(involve pi / lone pair e)
UV visible
Anthocyanin
Absorption of UV/vis by organic molecule and pigments
Less conjugatedsystem
↓
Less alternating single/double bond
↓
Absorb shorter wavelength (UV)
↓
Colourlesscompound
More conjugated system
↓
More alternatingsingle/double bond
↓
Absorb longer wavelength (visible)
↓
Colour compound
alternating double/single bond
More conjugation → More delocalization → Absorption in visible range
Extensive conjugation of double bond allow more delocalization of π elec
More conjugation → More delocalization → Less energy to excite electron → ↓ E lower ( absorb at visible region (colour )
How number of conjugation leads to colour formation from UV to visible?
BiologicalPigments(Anthocyanins)
Coloured – extensiveconjugation of electron
Alternatingsingle and double bond
Electron in pi orbitaldelocalized through single and double bond.
π elec excitedby absorbinglong wavelength in visible region

9. UV visible
Absorption of UV/vis by organic molecule and pigments
More conjugation → More delocalization → Absorption in visible range
Extensive conjugation of double bond allow more delocalization of π electron
More conjugation → More delocalization → Less energy to excite electron → ↓ E lower ( absorb visible region (colour )
How number of conjugation leads to colour formation from UV to visible?
More conjugation – splittingenergy less ∆E ↓ – wavelengthincrease (visible range)
Filled orbital
empty orbital
100 200 300 400 700nmWavelength λ
C – C C = C C = C – C = C C = C – C = C – C = C
∆E ↓with more conjugation
absorb from UV to visible
∆E ↓with more conjugation
Absorb at ↓ lower energy (↑ longer λ)
AbsorbUV – sunblock Absorb visible region – food dye (Azo dye)Acid/baseindicator

10. alternating double/single bond
CaroteneAnthocyanin Chlorophyll Heme (hemoglobin)
Wavelength - absorbed
Visible
light
Colour seen RED – RED reflect to eyes
- Blue absorb (complementary colour)
absorbed
RED
transmitted
Carotenoidsabsorb λ at 460 nm
BiologicalPigments(Anthocyanins)
Colour – extensiveconjugation ofelec.Alternatingsingle/double bond
π elec delocalizedthroughsingle/doublebond.
π elec excitedby absorbinglongwavelength in visible region
700 600 500 400

11. alternating double/single bond
CaroteneAnthocyanin Chlorophyll Heme (hemoglobin)
Wavelength - absorbed
Visible
light
Colour seen GREEN– GREEN reflect to eyes
- Red/Blue absorb (complementary colour)
absorbed
Green
transmitted
Chlorophyll absorb λ at 400 and 700nm
BiologicalPigments(Anthocyanins)
Colour – extensiveconjugation ofelec.Alternatingsingle/double bond
π elec delocalizedthroughsingle/doublebond.
π elec excitedby absorbinglongwavelength in visible region
700 600 500 400

12. CaroteneAnthocyanin Chlorophyll Heme (hemoglobin)
Wavelength - absorbed
Colour seen RED – RED reflect to eye
- Blue absorb
Anthrocyanin – acid base indicator
- absorb λ 550nm at pH 1 (acid)
Colour seen Yellow – yellow reflect to eye
- Blue absorb
Wavelength - absorbed
Anthrocyanin– acid base indicator
- absorb λ 470nm at pH 12 (alkali)
+ H+
+ OH-
Add acid
Add base
Changein numberOH gp
Change in numberconjugation
Absorbat diff wavelength
RED YELLOW
Numberconjugation increase
↓
Absorblonger wavelength
Numberconjugation decrease
↓
Absorb shorter wavelength
BiologicalPigments(Anthocyanins)
Colour– extensive conjugation ofelec.Alternating single/doublebond
π elec delocalized through single/double bond.
π elec excited by absorbing long wavelength in visible region

13. Anthocyanin
Wavelength - absorbed
Colour seen RED – RED reflect to eye
- Blue absorb
Anthrocyanin – acid base indicator
- absorb λ 550nm at pH 1 (acid)
Colour seen Yellow – yellow reflect to eye
- Blue absorb
Wavelength - absorbed
Anthrocyanin– acid base indicator
- absorb λ 470nm at pH 12 (alkali)
+ H+
+ OH-
Add acid
Add base
Change in numberOH gp
Change in numberconjugation
Absorb at diff wavelength
RED YELLOW
Numberconjugation increase
↓
Absorblonger wavelength
Numberconjugation decrease
↓
Absorb shorter wavelength
BiologicalPigments(Anthocyanins)
Anthrocyanins
Soluble – OH gp
C6C3C6 sys
Used as acid/baseindicator
Change in numberOH gp
Change in numberconjugation
Absorbdiff wavelength
Diff colour at diff pH
Colour – extensiveconjugation ofelec.Alternatingsingle/double bond
π elec delocalizedthroughsingle/doublebond.
π elec excitedby absorbinglongwavelength in visible region
Click here, diff colour diff pH
Click here, anthrocyaninchange colour at diff pH

14. Anthocyanins – used as acid/baseindicator
Identify λ max which correspondto max absorbanceat diff pH
and suggest colour in acid/basecondition.
pH Max Colour absorb Colour pigment
1 550 Green Red
12 475 Blue Yellow/orange
wavelength wavelength
Anthocyanins – used as acid/base indicator
Identify λ max which correspondto max absorbanceat diff pH
and suggest colour in acid/base condition.
pH Max Colour absorb Colour pigment
1 550 Green Red
7 350 None visible Colourless
Explain folowing observation
i. Carrot are boiled, little colouration inwater,when they are fried colour changeto orange
ii. Red cabbage is boiled, waterturn purple but when vinegar added colour change to red
Carotenoid are coloured due to extended π conjugation elec.(Non watersoluble long hydrocarbon chain)
In oil, they are soluble – produceda orange colour.
Colour due to anthrocyanin,watersolublecontainOH form H2 bondwith water. Colour changein diff pH (acid) due to diff numberof conjugationas its
protonated.
Non watersoluble – No colour in water
Carotenoids
ORANGE
Acid
RED
Base
YELLOW
Degree conjugation increase
↓
Absorb longer λ
Degree conjugation decrease
↓
Absorb shorter λ

15. Tetracene - Greater delocalization elec (Higher conjugation bond)
- Absorb longer wavelength – visible light (colour)
Organic compoundsshown anthracene and tetracene.
Predict with reference to conjugation double bond,which absorbvisible light (colour)
Carotene absorb light in blue/green region, so complementary
colour (red and orange) are transmitted
Anthracene Tetracene
Absorption spectrum ofcarotene was shown. Explain why carotene have colour.
Carotene
700 600 500 400
RED
Absorption spectrum of anthrocyaninis shown.
Explainwhat effect,the absorptionat 375 and 530 nm have on colour of anthrocyanin
At 375 nm - No effect, lies outside visible spectrum (UV region)
At 530 nm - Visible colour, red, complementary to blue-green
- Absorb green – Reflect Red
700 600 500 400 300 200
Anthocyanin
RED

16. Absorption of UV by organic molecules and chromophore
How Phenolphthalein indicator changes colour ?
Reason for colour change
• change in conjugation
• change in delocalization
Acidic
Colourless
Limiteddelocalization,only in benzene ring
Carbonsp3 – prevent delocalizationon whole sys
AbsorbUV region
Alkaline
Pink
Delocalization on whole sys
Extensive delocalization in 3 benzene ring
Carbon sp2 – allow delocalization
Absorb at visible region
Acid
colourless
Base
PINK
PinkColourless
sp3 – Prevent delocalization whole sys
Lower degree conjugation
sp2 – Allow delocalization whole sys
Higher degree conjugation
∆E energydiff ↑ higher
Less conjugation
Less delocalization
AbsorbUV region
∆E
∆E
∆E energydiff ↓ lower
More conjugation
More delocalization
Absorbvisible region