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