Abnormal hemoglobin

Changes in internal amino acidsHemolytic anemia

Changes on the surfaceHbSHbE

Changes stabilizing metHbMethemoglobinemia

Changes stabilizing T or R statesPolycythemia (R)

Cyanosis (T)1

Electron micrograph of deoxyHbS fibers spilling out of a ruptured erythrocyte. E6V mutation.

2

Locked in the T state3

4

Structure of the deoxyHbS fiber

5

There are no sickle cell aggregates in arteries

In the short time when blood passes through capillaries aggregates can form only if the blood is

moving slower than the aggregation time

Small changes in blood flow, O2 content, HbS concentration,

temperature will affect the sickling.

Origin of sickle cell crises

6

Mutations that inactivate hemoglobin

7

What is the role of the distal histidine?

Fe(II) + O2 Fe(III)-O2-

Fe(III)-O2- + H+ Fe(III) + HO2

Autoxidation

8

Mutations stabilizing the Fe(III) oxidation state of heme.

Result: MethemoglobinemiaCyanosis, brown blood

9

Hb Yakima: loss of H-bond that stabilizes TResult: lack of cooperativity, very high affinity for

oxygen Polycythemia (excess red blood cells)Hyperviscous blood, clotting

Ruddy complexion

Hb Kansas: loss of H-bond that stabilizes RResult: low cooperativity, low affinity for oxygen

PolycythemiaHyperviscous blood, clotting

Ruddy complexion

10

Properties of Enzymes

Function as catalysts only: play no role in the net rxn

Have no effect on equilibrium or ∆G

Lower the activation energy and thus affect kinetics

Generally have catalytic cofactors

Are usually highly substrate-specific

Are highly regulated

Often prevent more favorable chemistry from happening

11

12

∆G = ∆H - T∆S

Cell need a source of free energy

∆G’º = -RT ln Keq

Free energy depends on equilibrium constant

Keq = [P]/[S]

13

14

Keq = [G6P]/[G1P] = 19 mM/1 mM = 19

Glucose-1-phosphate

∆G’º = -RT ln Keq

At room temperature ∆G’º = -7.3 kJ/mol

Glucose-6-phosphate

15

16

Actual free energy depends of reactant and product concentrations

When ∆G = 0 this is equilibrium and

∆G’º = -RT ln Keq

This allows you to calculate actual ∆G’ in real conditions

∆G’ = ∆G’º + RT ln Keq

17

Enzymes don’t affect ∆G of the reaction

18

19

Enzymes affect rate by several mechanisms

1) Binding transition states2) Proximity effects3) Arresting atomic motions 4) Alter the solvent by excluding water

and changing pKa’s, use metal ions and protein side chains to alter electrostatics

5) Alter the substrate by forming transient covalent bonds

6) Using cofactors to change the chemistry

20

∆GB is energy of binding transition state by enzyme:

Major source of activation energy

21

Transition State Stabilization

R

R

CH2OH

COOH

R

R

C

O

O

R = -H or -CH3

Rate is 300x faster with CH3

22

23

Proximity and Orientation Effects

Reactants must come together with the proper spatial

relationship

24

NH

N

H3C C

O

O NO2

NH

N+

H3C

C

O

-O NO2

NH

N

C

O

O NO2

NH

N+

-O NO2C

O

24 fold enhancement of rate

Proximity - small effect

25

Orientation - large effect

Molecules react most readily only if their molecular orbitals

are oriented properly

26

The geometry of an SN2 reaction.

Deviation by 10º will result in 100 fold rate dimunition27

Elimination of motion/entropy reduction

Enzymes immobilize substrates

28

Exclusion of water changing electrostatics

+H3N

C

C

O

O-

CH2H

NNH

C

O

-O

CH2OH

C

O

OH

29

Metal Ions alter electrostatics

Metalloenzymes(Fe, Zn, Cu, Mn, Co, Ni, Na, K, Ca, Mg)

Substrate binding and orientationShielding of negative charges

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Covalent catalysis

31

Enzyme active sites are designed for specific substrates

32

Geometric specificity

Many ADH enzymes accept different size substrates

Few enzymes are absolutely specific

CH3OHCH3CH2OH

33

R

C

O

N

H

R'

R

C

O

O

R'

R

C

O

O- +H3N

R'

R

C

O

O- HO

R'

Chymotrypsin catalyzes both ester and amide hydrolysis

34

C Hpro-RHpro-S

OH

CH3

Pro-chiral ethanol

CH3CH2OH + NAD+

YADHCH3CHO + NADH + H+

Stereospecificity

35

C DD

OH

CH3

N+

R

C

O

NH2

H

N

R

C

O

NH2

D

C

O

H3C D

H

NAD+ NADD

YADH

R isomer

36

C DH

OH

CH3

N+

R

C

O

NH2

H

N

R

C

O

NH2

D

C

O

H3C H

H

NAD+ NADD

H+

R isomer

R isomer

37

C HD

OH

CH3

N+

R

C

O

NH2

H

N

R

C

O

NH2

H

C

O

H3C D

H

NAD+ NADH

H+

S isomer

Non-chiral

38

CH3

HproS O

HproR

N+

HH

R

H C

H

O

H2N

si-side

re-side

C-

HH

R

H C O

H2N

Hpro-S

Hpro-R

H

B

N+

HH

R

H C

H

O

H2Nsi-side

re-side

C-

HH

R

H C O

H2N

Hpro-S

Hpro-R

H3C

O H BHproS

39

Enzymes have coenzymes and cofactors

Organic

40

Enzymes have coenzymes and cofactorsInorganic

Cu2+, Fe2+, Mn2+, Ni2+, Mo4+ Electron transferZn2+, Ni2+, Fe3+, Mn2+, Mg2+, K+ Charge stabilization 41

Vitamins That Are Coenzyme Precursors.

Zn2+ Acrodermatitis enteropathicaCu2+ Menkes diseaseFe2+ Anemia 42

Enzyme activity is regulated

1. Gene transcription2. mRNA translation3. Enzyme localization4. Enzyme activity

43

Allosteric Regulation

Effectors/Modulators

Homotropic/Heterotropic

Positive/Negative

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45

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OC

NH2

OPO32-

O

C

O-

CH2

C

COO-+H3N

H+NH2

C

NH

CH

CH2

C

O COO-

O-

O

Aspartate transcarbamolyase

47

OC

NH2

OPO32-

O

C

O-

CH2

C

COO-+H3N

H+NH2

C

NH

CH

CH2

C

O COO-

O-

O

NH2

C

NH

CH

CH2

C

O COO-

O-

O

HN

NH

O COO-

O

Orotate: precursor for pyrimidines

48

The rate of the reaction catalyzed by ATCase as a function of aspartate

concentration 49

Schematic representation of the pyrimidine biosynthesis pathway.

50

∆Gº’ values for sequential reactions are additive

(1) A ---> B ∆Gº’1

(2) B ---> C ∆Gº’2

Sum: A ---> C ∆Gº’1 + ∆Gº’2

Enzymes can couple endergonic reactions with exergonic ones to make

them go spontaneously

51

Enter ATP

52

53

54

Other ‘high energy’ phosphate compounds

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57

58

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