Option B Enzyme Kinetics, Pigment and Anthrocyanin electron conjugation
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Transcript of Option B Enzyme Kinetics, Pigment and Anthrocyanin electron conjugation
Evaluate specificity of enzyme for substrate Low Km – High affinity High Km – Low affinity
SK
SVv
m
][max
Enzyme kinetics
Michaelis Menten eqn
Enzyme Enzyme/substrate complex Product
Rate
Sub conc
Max rate velocity
2
maxVKm
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
SVv
m
][max
Low [S] Km >[S]
High [S] Km <[S]
][
][
max
max
SK
Vv
K
SVv
m
m
(1st order) rate prop to [S] All active site available
max
max
max
][
][
Vv
SS
Vv
SK
SVv
m
(zero order) to [S] All active site saturated
constant
Evaluate specificity of enzyme for substrate Low Km – High affinity High Km – Low affinity
SK
SVv
m
][max
Enzyme kinetics
Michaelis Menten eqn
Enzyme Enzyme/substrate complex Product
Rate
Sub conc
Max rate velocity
2
maxVKm
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
Kefficiencycatalytic .
mcat KK
Kcat = turnover number- max sub convert to product per second (Enzyme saturated)
Click here view KM Click here Michaelis Menten
Competitive Inhibitor
Rate
Sub conc
Max rate velocity
Km = [S] when rate is half Vmax
Rate
SK
SVv
m
][max
Low [S] Km > [S]
High [S] Km < [S]
][
][
max
max
SK
Vv
K
SVv
m
m
(1st order) rate prop to [S] All active site available
max
max
max
][
][
Vv
SS
Vv
SK
SVv
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 inhibitor substrate
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
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
V max – Lower ↓ - Enzyme unavailable Don’t alter active site – no effect on Km
Alter conformational change enzyme NOT substrate binding (affinity) Km - Unchange- Enzyme affinity unchanged
V max – Unchanged – High [S} will reduced inhibition Compete for same active site - substrate binding (affinity) lower ↓ Km - Change- Enzyme affinity lower ↓
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
V max – Lower ↓ - Enzyme unavailable Don’t alter active site – no effect on Km Km - Unchange- Enz affinity unchanged
Km same
2
maxVKm
Rate
Sub conc
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 enzymes and inorganic catalyst
Enzyme Catalyst
Similarity Both increase rate
Both lower activation energy
No effect on yield
Differences Protein Not protein
Show saturation kinetic (hyperbolic curve)
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
maxVKm
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 empty orbital
π electron
Absorb UV to excite π/lone pair e to higher empty orbital
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
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 higher empty orbital
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 transition from bonding to antibonding orbital
(involve pi / lone pair e)
UV visible
Organic molecules/chromophores
Biological Pigments (Anthocyanins) Coloured – extensive conjugation of electron
Alternating single and double bond Electron in pi orbital delocalized through single and double bond.
π elec excited by absorbing long wavelength in visible region
Anthocyanin
Chlorophyll
absorb absorb absorb absorb
Higher empty orbital
Absorb radiation to excite delocalized e to empty orbital
Filled orbital
empty orbital
Carotene
Colourless – Absorption in UV range Electronic transition from bonding to antibonding orbital
(involve pi / lone pair e)
UV visible
Anthocyanin
Absorption of UV/vis by organic molecule and pigments
Less conjugated system ↓
Less alternating single/double bond ↓
Absorb shorter wavelength (UV) ↓
Colourless compound
More conjugated system ↓
More alternating single/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?
Biological Pigments (Anthocyanins) Coloured – extensive conjugation of electron
Alternating single and double bond Electron in pi orbital delocalized through single and double bond.
π elec excited by absorbing long wavelength in visible region
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 – splitting energy less ∆E ↓ – wavelength increase (visible range)
Filled orbital
empty orbital
100 200 300 400 700nm
Wavelength 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)
Absorb UV – sunblock Absorb visible region – food dye (Azo dye) Acid/base indicator
alternating double/single bond
Carotene Anthocyanin Chlorophyll Heme (hemoglobin)
Wavelength - absorbed
Visible light
Colour seen RED – RED reflect to eyes - Blue absorb (complementary colour)
absorbed
RED
transmitted
Carotenoids absorb λ at 460 nm
Biological Pigments (Anthocyanins)
Colour – extensive conjugation of elec. Alternating single/double bond π elec delocalized through single/ double bond.
π elec excited by absorbing long wavelength in visible region
700 600 500 400
alternating double/single bond
Carotene Anthocyanin 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
Biological Pigments (Anthocyanins)
Colour – extensive conjugation of elec. Alternating single/double bond π elec delocalized through single/ double bond.
π elec excited by absorbing long wavelength in visible region
700 600 500 400
Carotene Anthocyanin 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
Change in number OH gp Change in number conjugation Absorb at diff wavelength
RED YELLOW
Number conjugation increase ↓
Absorb longer wavelength
Number conjugation decrease ↓
Absorb shorter wavelength
Biological Pigments (Anthocyanins)
Colour – extensive conjugation of elec. Alternating single/double bond π elec delocalized through single/ double bond.
π elec excited by absorbing long wavelength in visible region
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 number OH gp Change in number conjugation Absorb at diff wavelength
RED YELLOW
Number conjugation increase ↓
Absorb longer wavelength
Number conjugation decrease ↓
Absorb shorter wavelength
Biological Pigments (Anthocyanins)
Anthrocyanins Soluble – OH gp C6C3C6 sys Used as acid/base indicator Change in number OH gp Change in number conjugation Absorb diff wavelength Diff colour at diff pH
Colour – extensive conjugation of elec. Alternating single/double bond π elec delocalized through single/ double bond.
π elec excited by absorbing long wavelength in visible region
Click here, diff colour diff pH
Click here, anthrocyanin change colour at diff pH
Anthocyanins – used as acid/base indicator Identify λ max which correspond to max absorbance at diff pH
and suggest colour in acid/base condition.
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 correspond to max absorbance at 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 in water, when they are fried colour change to orange ii. Red cabbage is boiled, water turn purple but when vinegar added colour change to red
Carotenoid are coloured due to extended π conjugation elec. (Non water soluble long hydrocarbon chain) In oil, they are soluble – produced a orange colour. Colour due to anthrocyanin, water soluble contain OH form H2 bond with water. Colour change in diff pH (acid) due to diff number of conjugation as its protonated.
Non water soluble – No colour in water Carotenoids
ORANGE
Acid RED
Base YELLOW
Degree conjugation increase ↓
Absorb longer λ
Degree conjugation decrease ↓
Absorb shorter λ
Tetracene - Greater delocalization elec (Higher conjugation bond) - Absorb longer wavelength – visible light (colour)
Organic compounds shown anthracene and tetracene. Predict with reference to conjugation double bond, which absorb visible light (colour)
Carotene absorb light in blue/green region, so complementary colour (red and orange) are transmitted
Anthracene Tetracene
Absorption spectrum of carotene was shown. Explain why carotene have colour.
Carotene
700 600 500 400
RED
Absorption spectrum of anthrocyanin is shown. Explain what effect, the absorption at 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
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
Limited delocalization, only in benzene ring Carbon sp3 – prevent delocalization on whole sys
Absorb UV 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
Pink Colourless
sp3 – Prevent delocalization whole sys Lower degree conjugation
sp2 – Allow delocalization whole sys Higher degree conjugation
∆E energy diff ↑ higher Less conjugation
Less delocalization Absorb UV region
∆E
∆E
∆E energy diff ↓ lower More conjugation
More delocalization Absorb visible region