Abnormal hemoglobin Changes in internal amino acids Hemolytic anemia Changes on the surface HbS HbE Changes stabilizing metHb Methemoglobinemia Changes stabilizing T or R states Polycythemia (R) Cyanosis (T) 1

Electron micrograph of deoxyHbS fibers spilling out of a ruptured erythrocyte. E6V mutation. 2

Locked in the T state 3

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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, O 2 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) + O 2 Fe(III)-O H + Fe(III) + HO 2 Autoxidation 8

Mutations stabilizing the Fe(III) oxidation state of heme. Result: Methemoglobinemia Cyanosis, brown blood 9

Hb Yakima: loss of H-bond that stabilizes T Result: 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 R Result: low cooperativity, low affinity for oxygen Polycythemia Hyperviscous 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

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∆G = ∆H - T∆S Cell need a source of free energy ∆G’º = -RT ln K eq Free energy depends on equilibrium constant K eq = [P]/[S] 13

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K eq = [G6P]/[G1P] = 19 mM/1 mM = 19 Glucose-1-phosphate ∆G’º = -RT ln K eq At room temperature ∆G’º = -7.3 kJ/mol Glucose-6-phosphate 15

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Actual free energy depends of reactant and product concentrations When ∆G = 0 this is equilibrium and ∆G’º = -RT ln K eq This allows you to calculate actual ∆G’ in real conditions ∆G’ = ∆G’º + RT ln K eq 17

Enzymes don’t affect ∆G of the reaction 18

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Enzymes affect rate by several mechanisms 1)Binding transition states 2)Proximity effects 3)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

∆G B is energy of binding transition state by enzyme: Major source of activation energy 21

Transition State Stabilization R = -H or -CH 3 Rate is 300x faster with CH 3 22

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Proximity and Orientation Effects Reactants must come together with the proper spatial relationship 24

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 S N 2 reaction. Deviation by 10º will result in 100 fold rate dimunition 27

Elimination of motion/entropy reduction Enzymes immobilize substrates 28

Exclusion of water changing electrostatics 29

Metal Ions alter electrostatics Metalloenzymes (Fe, Zn, Cu, Mn, Co, Ni, Na, K, Ca, Mg) Substrate binding and orientation Shielding of negative charges 30

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 CH 3 OH CH 3 CH 2 OH 33

Chymotrypsin catalyzes both ester and amide hydrolysis 34

Pro-chiral ethanol CH 3 CH 2 OH + NAD + YADH CH 3 CHO + NADH + H + Stereospecificity 35

YADH R isomer 36

R isomer 37

S isomer Non-chiral 38

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Enzymes have coenzymes and cofactors Organic 40

Enzymes have coenzymes and cofactors Inorganic Cu 2+, Fe 2+, Mn 2+, Ni 2+, Mo 4+ Electron transfer Zn 2+, Ni 2+, Fe 3+, Mn 2+, Mg 2+, K + Charge stabilization 41

Vitamins That Are Coenzyme Precursors. Zn 2+ Acrodermatitis enteropathica Cu 2+ Menkes disease Fe 2+ Anemia 42

Enzyme activity is regulated 1.Gene transcription 2.mRNA translation 3.Enzyme localization 4.Enzyme activity 43

Allosteric Regulation Effectors/Modulators Homotropic/Heterotropic Positive/Negative 44

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Aspartate transcarbamolyase 47

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

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Other ‘high energy’ phosphate compounds 55

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