Biochem Test 2

Front 1

Hemoglobin Evolution - atmospheric considerations

Back 1

atmosphere changed from reducing to oxidizing 2 billion years ago

Fe+2 oxidized to Fe+3 - mass extinction

electron-deficient properties of O2 harnesed to increase oxidative metabolism efficiency and diversity

Front 2

Globin secondary structure

Back 2

alpha-helices (78%), remainder in loops joining the helices

no beta sheets

Front 3

Hemoglobin Evolution - phylogeny

Back 3

single globin chain -> two chains via gene duplication (alpha and beta) during higher fish evolution - marine worms, insects, primitive fish have single globin chain

alpha and bega genes translocated to different chromosomes in birds and mammals, but on same chromosome in frog Xenopus

in mammals alpha and beta chains duplicated and diversified

Front 4

Hemoglobin α and β chains in mammals

Back 4

beta chain has greatest effect - beta chains have different 02 binding affinities.

beta diversified into fetal (ε,γ) and adult (β,δ) forms.

tetramers of 2α2β required for functionality - β homotetramer possible if β overexpressed but it is not functional

fetal beta forms (epsilon and gamma) have higher 02 affinity, allowing transfer from maternal to fetal complexes.

alpha2delta2 heterotetramer a minor component of primate adult hemoglobin

Front 5

Ligand

Back 5

molecule that reversibly binds to an protein. Removal of water from the binding site and the ligand (desolvation) results in an initial activation energy cost.

ligand and protein have binding site complementarity which equates to high specificity

Front 6

Ligand binding

Back 6

concentration-dependent, high concentration increases likelihood of collision between specific protein and ligand allowing maximum binding interactions

electrostatic charge complementarity can also direct binding.

binding coupled to conformational change in protein

Front 7

dissociation constant

Back 7

Kd = ([P][L]) / [PL]

P=protein, L=ligand

Smaller Kd => greater affinity

Kd is [L] at which 50% of binding sites are occupied

Front 8

θ

Back 8

fraction of binding sites occupied by ligand

θ = (binding sites occupied)/(total binding sites)

θ = [PL] / (]PL] - [P])

θ = [L] / ([L] + Kd)

[L] = (θ Kd) / (1 - θ)

Front 9

biological O2 binding

Back 9

Reduced transition metals can bind O2 - Fe2+ and Cu+, but not Fe3+ and Cu2+

Fe2+ spontaneously oxidizes with O2 and hydroxyl radicals are formed

four N form porphyrin coordinate with Fe2+ and stabilize the 2+ state ofer the 3+

2 free hemes + O2 = irreversible oxidation to Fe3+

Heme sequestered in protein with His (F8) coordinated to one of the two remaining sites - stabilizes the Fe2+ by donating e- and leaves only one binding site for O2

O2 binds reversibly to heme so sequestered and there is no change in Fe oxidation state

Front 10

Myoglobin (Mb)

Back 10

single chain globin

most abundant in muscle, aids O2 transport through tissue

Diving mammals have high Mb in muscles which serves as an O2 store also.

Front 11

Myoglobin and O2 binding

Back 11

partial pressure of O2 measured (dissolved O2 is difficult to measure)

θ = pO2 / (pO2 + P50)

P50 = partial pressure of O2 at which half of ligand sites are occupied

Front 12

CO

Back 12

binds free heme with 20000 X higher affinity than O2

heme bound to Mb or Hb CO affinity 200 (Mb) or 250 (Hb) X greater than O2 - CO cannot form linear conformation due to steric hinderance due to a His residue

The His residue stabilizes the bent O2 binding

Front 13

CO poisoning

Back 13

more than 50% of worldwide poisoning deaths

CO bound to 1 or 2 Hb subunits increases O2 affinity at other sites - does not release O2 at the tissues

Front 14

Siclke cell anemia

Back 14

point mutation - Glu to Val - homozygotes show symptoms

beta chain conformation not significantly changed

hydrophobic residue placed on surface

The Val fits into hydrophobic pocket of another Hb when deoxygenated

deoxyHbS polymerizes into fibers during anoxic conditions - physical exertion

Hb content of sickle-cell blood is ~50% that of normal individuals - sickle cells are fragile and rupture easily

sickle cells can block small capillaries

heterozygotes have slight resistance to malaria

Front 15

Hemoglobin structure

Back 15

4 subunits (2 alpha, 2 beta)

subunits have globin tertiary fold with a heme prosthetic group - each Hb can bind up to 4 O2

alpha and beta subunits are adjacent and have more interactions than between alpha-alpha or beta-beta which are opposite

Front 16

Hemoglobin states

Back 16

T state - more stable in absence of O2,
R state - higher O2 affinity, O2 binding stabilizes R state

Transition does not cause large change in individual subunits, but extensive changes at the alpha-beta interfaces - T -> R disrupts some ionic interactions, while some new ones are formed.

α-β interface has >30 interactions, most hydrophobic, and some ionic (salt bridges or ion pairs)

Front 17

inactive forms of enzymes

Back 17

proenzymes or zymogens

common for proteases, e.g. chymotrypsin from chymotrypsinogen by cleavage of short N-terminus peptide upon entry into digestive tract

Front 18

proteases

Back 18

degrade proteins

inactivated by inhibitor proteins that are tightly bound to the active site

e.g. pancreatic trypsin inhibitor

Front 19

proteosome

Back 19

in eukaryotes, degrades proteins

constitutes about 1% of cellular protein

consists of 20S core and two 19S caps

some core subunits are proteases with active sites lining cylinder

19S caps composed of ca. 18 subunits, ~6 of which hydrolyze ATP

proteins targeted for transport to proteasome by attachment of chains of ubiquitin

Front 20

ubiquitin

Back 20

76 a.a. protein

C-terminus covalently linked to amino group on side chain of K (isopeptidic bond)

Chains of at least 4 ubiquitins are needed to target proteins to the proteasome

C-terminus also attaches to K of another ubiquitin to form chains

Front 21

ubiquitination - associated enzymes

Back 21

E1 - ubiquitin-activating enzyme
E2 - ubiquitin-conjugating enzyme (~30 in mammals)
E3 - ubiquitin ligase (>200 in mammals)

E2 and E3 act as a complex

E3 is key, recognizes different degradation signals on proteins

ca. 300 E2/E3 combinations found in mammals

Front 22

ubiquitin activation

Back 22

E1 forms high-energy thioester bond between a C residue and the C-terminus of ubiquitin via an ATP coupled reaction.

E1 then transfers ubiquitin to a C residue on the E2 of an activated E2/E3 complex

Front 23

ubiquitination - targeting

Back 23

E3 recognizes specific degradation signals.

This allows E2 to transfer ubiquitin to a K residue on the target

The proteosome 19S cap recognizes the polyubiquitin chain.

Ubiquitin is cleaved from target protein and re-used

Front 24

ubiquitination - ligase complex activation

Back 24

E2/E3 complex activated by:

-phosphorylation

-allosteric transition due to ligand binding

-allosteric transition due to protein subunit addition

These processes can also occur on the target protein which helps mark it for ubiquitination

Front 25

ubiquitination - N-terminal targeting

Back 25

the N-terminal residue can target protein for degradation - identity correlated to half-life in the cell in a clock-like manner

Stabilizing residues are: M, G, A, S, T, V. these give a half-life of >20 hours.

Destabilizing residues (some others others) give half-lives from 2 to 30 minutes

Front 26

Metabolism - general

Back 26

catabolism - breakdown of energy-containing compounds. Energy stored in high-energy or reduced compounds, e.g. ATP and NADH

anabolism - synthesis of complex molecules - requires free energy

catabolism converges on cyclic pathway, while anabolism diverges to create wide variety of compounds

Front 27

metabolism - categories of reactions

Back 27

4 major categories:

1. Oxidation/reductio

2. Reactions that make or break C-C bonds.

3. Internal rearrangements (isomerizations like trans to cis P) and eliminations (e.g. dehydration)

4. Group transfers: acyl, glycosyl, phosphoryl, etc.

Front 28

Group transfer

Back 28

via a Sn2 mechanism with tetrahedral intermediate

Phosphoryl transfer - from ATP is the main way chemical energy is transferred during catalysis

Front 29

ATP Hydrolysis - large negative free energy change

Back 29

1. Relief of electrostatic repulsion between the 4 negative charges by separation

2. Pi is resonance stabilized.

3. ADP(2-) immediately ionizes releasing H+ at ~ph =7

4. Greater degree of solvation relative to ATP

Front 30

non-ATP chemical energy stores

Back 30

PEP3- to pyruvate- and Pi

Acetyl-CoA to acetic acid to acetate ion

Phosphocreatine to creatine

Front 31

ATP as energy currency

Back 31

ATP is intermediate in phosphorylation potential - it can transfer phosphoryl groups from higher-energy phosphate compounds from catalysis to other compounds, such as glucose

ATP is kinetically stable - does not spontaneously transfer Pi groups, requires enzyme

Front 32

ATP group transfer mechanism

Back 32

usually written as 1-step hydrolysis, but generally achieved by 2-step covalent group transfer

processes that involve direct hydrolysis of ATP usually linked to mechanical motion e.g. protein comformational changes, muscle contractions, movement of protein complexes along DNA

Front 33

ATP structure

Back 33

Front 34

nucleophilic attack on ATP

Back 34

3 positions for attack:
γ - phosphoryl transfer; R-Pi + ADP
β - pyrophosphoryl transfer; R-P-Pi + AMP
α - adenylyl transfer; R-P-Adn + PPi

pyrophosphoryl transfer releases greater G than phosphoryl - used for more unfavorable reactions e.g. ubiquitin charging

Front 35

adenylyl group transfer

Back 35

adenylyl group = 5'AMP

generates pyrophosphate (PPi) which is further hydrolyzed to release more free energy by inorganic pyrophosphatase

coupled to very unfavorable reactions such as fatty acid activation

Front 36

fatty acid activation

Back 36

highly unfavorable, driven by adenylylation

adenylyl transferd to carboxyl group of fatty acid, then coenzyme A displaces the adenylyl group to form thioester

Front 37

metabolic regulation

Back 37

12% of human genes encode regulatory proteins

maintaining constant [ATP] is second most important task for cells

typical [ATP] = 5mM in cells

typical ATP-consuming enzymes have Km ~ .0 to 1 mM

if [ATP] decreases, many reactions would not occur and cell would die

[ATP]/[ADP] is critical

Front 38

[AMP] as indicator

Back 38

[AMP] usually < 0.1mM

[ADP] usually ~ 1.0mM

Adenylate kinase catalyzes:
2ADP -> AMP + ATP

drop in [ATP] leads to greater relative increase in [AMP] than [ADP]

many reactions are regulated by [AMP]

Front 39

induced fit

Back 39

protein conformational changes upon initial binding with ligand to optimize interactions

Front 40

O2 binding to Hemoglobin

Back 40

-can bind to both states, but R has higher affinity.

-binds in the distal pocket, which stabilizes the R-state

T-state has porphyrin ring slightly bent toward the proximal His ligand - on binding O2, porphyrin ring becomes more planar

Front 41

proximal and distal pockets

Back 41

proximal - binding pocket has porphyrin ring bowed toward the proximal His. Occurs in the T state.

distal - pocket more open, porphyrin ring more planar, O2 (or other ligand/solvent) can bind.

Front 42

Hemoglobin cooperative binding

Back 42

transition from low to high affinity states.

Transition from T to R state is positively cooperative - each O2 that binds, raises affinity in the remaining subunits.

produces sigmoidal curve on a plot of θ vs. pO2. (non-cooperative binding produces hyperbolic curve)

Front 43

Allostery

Back 43

binding of a ligand to one site causes a conformational change that alters affinity at another site.

positive and negative cooperativity

heterotropic - different ligands at each site

homotropic - same ligands bind at each site

Front 44

allostery models

Back 44

symmetry model - all sites either in high or low affinity states. Ligand binding increases high affinity state and alters equilibrium between states. All or nothing change in quaternary structure.

sequential model - individual sites can be in different states. Ligand binding increases proportion of sites in high-affinity state.

Front 45

cooperative binding equations

Back 45

for n-sites:

Kd = ([P][L]^n) / [PL]

θ = ([L]^n) / ([L]^n = Kd)

Front 46

Hill equation

Back 46

calculates the relative saturation

θ / (1 - θ) = ([L]^n) / Kd

Front 47

Hill plots

Back 47

log(θ / (1 - θ)) vs. log[L]

[L] = conc. of free L

slope = n

Front 48

Hill coefficient

Back 48

nsubh

measure of degree of cooperativity

n ≠ # of binding sites

* n is always less than # binding sites *

Front 49

hemoglobin and myoglobin Hill plots

Back 49

myoglobin n = 1

hemoglobin T and R states n=1

hemoglobin in cooperative range n ≈ 3

hemoglobin - linear with slope = 1

myoglobin - sigmoidal with two asymptotes

Front 50

Bohr effect - hemoglobin in tissues

Back 50

CO2 from metabolism converted to carbonic acid (H2CO3) by anhydrase - then dissociates to lower pH. Tissue pH ≈ 7.2.

This protonates some of the residues involved in ionic interactions between the subunits which favors O2 release

A His residue on the β subunit forms ionic pair with an Asp on the α subunit. This gives the His an abnormally high pKa and the ion pair stabilizes the T state.

O2 released into the tissues, CO2 (carbonic acid) taken into erythrocytes.


Proton binding antagonistic to O2 binding

Front 51

Bohr effect - hemoglobin in lungs

Back 51

lung pH = 7.6. The R-state does not have this ion pair and the His pKa drops back to 6 and is mostly unprotonated.

High pO2 in lungs favors O2 binding, stabilizing the R-state.

Released protons combine with bicarbonate to form CO2 via carbonic anhydrase. CO2 exhaled

O2 binding antagonistic to CO2 (proton) binding.

Front 52

BPG

Back 52

2,3-bisphosphoglycerate

formed in erythrocytes

heterotropic allosteric modulator of Hb

Front 53

BPG and Hb

Back 53

single BPG binds to positively charged cavity between subunits in T-state. This makes the transition to R more difficult and lowers O2 affinity.

little effect on O2 bound in the lungs, large effect on release in the tissues.

maintains ≈ 40% O2 delivery to tissues at different altitudes (different pO2)

without PBG Hb's high O2 affinity would prevent release in the tissues

Fetal Hb (HbF) has lower BPG affinity and thus higher O2 affinity than adult Hb (HbA). This allows O2 transfer from maternal to fetal Hb across the placenta.

Front 54

catalyst

Back 54

alters rate (but not equilibrium) of a reaction by many orders of magnitude

following a complete cycle, original state is returned

Front 55

catalytic power

Back 55

rate of catalyzed reaction over rate of uncatalyzed reaction

for enzymes usually 10e5 to 10e17

Front 56

6 classes of enzymes

Back 56

1. oxidoreductases - xfer of electrons (hydride ions or H atoms)

2. transferases - group transfer

3. hydrolases - hydrolysis reactions (transfer of groups to water)

4. lyases - addition of groups to double bonds / formation of double bonds by removal of groups

5. isomerases - internal rearrangements

6. ligases - formation of C-C, C-O, C-S, and C-N bonds by condensations coupled with ATP cleavage

Front 57

enzymes - general

Back 57

most are proteins, few are RNA (ribozymes)

some require cofactors such as metal ions

some require coenzymes such as NAD+ or FAD

prosthetic groups are coenzymes or cofactors that are covalent bonded to enzyme

holoenzyme = cofactor/coenzyme + apoenzyme/apoprotein

complementarity of binding sites gives high specificity - usually stereospecific

no effect on ΔG or Keq

Front 58

Keq and free energy

Back 58

10fold increase of K'eq ≈ 6 kJ/mol of stabiliztion or addition of ca. 1.5 hydrogen bonds per molecule

Front 59

enzymes and equilibria

Back 59

enzymes speed up both forward and backward reactions equally (think of carbonic anhydrase in the tissues vs the lungs)

overall product/reactant ratio of multiple sequential reactions influenced by individual Keq - coupling an unfavorable to a favorable reaction can result in product accumulation

Front 60

enzyme kinetics

Back 60

various distinct reaction intermediates formed with enzyme

most strained structure between intermediates is the transition state- decay into P or S equally likely at this point

enzymes lower energy barrier

enzymes stabilize particular transition states

Front 61

reaction intermediate

Back 61

has a finite lifetime - longer than a molecular vibration

Front 62

transition state

Back 62

transient, not possible to isolate

Front 63

reaction rates

Back 63

determined by concentration of reactants and rate constant k

unimolecular reaction: V = k[S] ; k is first order with units of s-1

bimolecular reaction: V = k[S][Y] ; k is second-order with units of M-1s-1

k = (kT/h)e^(-ΔG‡/RT)

Front 64

ΔG‡

Back 64

transition state energy barrier

contributing factors:

1. reduction of entropy required to orient 2 molecules correctly

2. removal of solvation shell from reactants

3. distortion or strain during reactions

4. proper alignment of catalytic groups

Front 65

Michaelis-Mente Equation

Back 65

V0 = kcat[ES]

Front 66

Km

Back 66

Michaelis constant
=[S] at which V0 = 0.5 Vmax

Front 67

Lineweaver-Burk plot

Back 67

double-reciprocal plot
1/Vmax = y-intercept
-1/Km = x-intercept

Front 68

bisubstrate reactions

Back 68

sequential and ping-pong

2 substrates going to two products, most common are transferase reactions

Front 69

sequential reaction

Back 69

both substrates bind to form ternary complex.

Double reciprocal plot with several lines for different [S] - lines intersect.

Front 70

ping-pong reaction

Back 70

double-replacement

no ternary complex formed

substrate 1 binds and leaves as product 2 - modifies enzyme

substrate 2 binds and leaves as product 2 - restores enzyme

Double reciprocal plot with lines for different [S] - lines parallel

Front 71

inhibition

Back 71

inhibit catalytic activity through binding

competitive or non competitive

Front 72

competitive inhibition

Back 72

know equation

lines intersect in double reciprocal plot at the y-intercept (1/Vmax) - changes Km, but not Vmax

competitive inhibitor binds to same active site - best are analogs of the transition state

Front 73

uncompetitive inhibition

Back 73

binds to the ES complex

often in multisubstrate reactions - often resembles second substrate (competitive with respect to second substrate)

lines parallel on double-reciprocal plot - decreases Vmax and Km

Front 74

mixed inhibition

Back 74

binds away from the substrate binding site

can bind to both free enzyme and complex

can alter binding affinity for substrate

Front 75

noncompetetive inhibition

Back 75

special case of mixed

does not alter substrate affinity, but prevents the reaction from occuring

double-reciprocal - lines intersect at x-axis - Km not affected

Front 76

reversible inhibition

Back 76

know equation

simplifies to competitive and uncompetitive equations when α' and α = 1

Front 77

irreversible inhibition

Back 77

covalent binding

suicide inhibition - undergoes first setps of enzymatic reaction, but instead of forming product, forms very reactive compound that binds to enzyme covalently.

Front 78

general acid-base catalysis

Back 78

transfer of proton (to group other than water) to stabilize intermediates

Front 79

specific acid-base catalysis

Back 79

transfer of proton to water to stabilize interiediates

Front 80

covalent catalysis

Back 80

catalyst has nucleophilic group

Front 81

metal ion catalysis

Back 81

-can help to orient substrate

-can participate in redox through changes in oxidation state

-can stabilize charged intermediates or transition states

Front 82

serine proteases

Back 82

catalyzes polypepptide backbone - yields smaller polypeptides

-have unusually reactive seine residue - all use same mechanism

trypsin, chumotrypsin, elastase (divergent evolution)

other families by convergent evolution

-also esterases (in addition to amidases)

Front 83

esterase activity of serine proteases

Back 83

burst phase - formation of acyl-enzyme intermediate

steady state phase - slower hydrolysis of acyl-enzyme intermediate

ping-pong mechanisom

Front 84

catalytic residues of chymotrypsin

Back 84

Ser195, His57, Asp102 - catalytic triad

Front 85

peptidoglycan

Back 85

NAG-NAM disaccharide repeat linked to tetrapeptide

tetrapeptide has 2 L-a.as

both saccharides are pyranoses (6-membered rings)

NAM has lactyl group on C3

Front 86

HEWL

Back 86

hen egg white lysozyme

NAM can only fit in sites B,D,F

cuts between D and E

D must be in half-chair conformation