Biomolecular Chemistry - Exam 1

Front 1

Competitive Inhibition - Lineweaver Burke Equation

Back 1

1/v=(1+[I/Ki])*(Km/Vmax)*(1/[S]) + 1/Vmax

Front 2

Non-Competitive Inhibition - Lineweaver Burke Equation

Back 2

1/v=(1+[I/Ki])*(Km/Vmax)*(1/[S]) + (1+[I/Ki])*1/Vmax

Front 3

Uncompetitive Inhibition - Lineweaver Burke Equation

Back 3

1/v=(Km/Vmax)*(1/[S]) + (1+[I/Ki])*1/Vmax

Front 4

No inhibitor - Lineweaver Burke Equation

Back 4

1/v=Km/Vmax)*(1/[S]) + 1/Vmax

Front 5

No inhibitor - Michaelis Menten Equation

Back 5

v=Vmax*[S]/(Km+[S])

Front 6

pK for alpha-carboxyl group (free)

Back 6

3

Front 7

pK for beta-carboxyl group

Back 7

4

Front 8

pK for imidazole

Back 8

6

Front 9

pK for sulfhydryl

Back 9

8

Front 10

pK for primary alpha-amino (free)

Back 10

8

Front 11

pK for epsilon amino, or phenolic hydroxyl

Back 11

10

Front 12

pK for secondary alpha amino (free)

Back 12

9

Front 13

pK for guanido

Back 13

12

Front 14

Amino acids with carboxyl groups

Back 14

Glutamate, Aspartate

Front 15

Amino acids with aromatic side groups

Back 15

Tryptophan and Phenylalanine

Front 16

What happens to a R group at pH above it's pk?

Back 16

R group will be less protonated

Front 17

What happens to an R group at a pH equal to it's pK?

Back 17

50% of the R groups will be protonated, and 50% will be unprotonated

Front 18

What happens to an R group at a pH below its pK?

Back 18

R group will be more protonated

Front 19

Enzymes alter the ____ energy of a reaction, no its change in ____ energy.

Back 19

They alter the ACTIVATION energy, and do not change FREE energy.

Front 20

How does high substrate concentration affect irreversible inhibitors/inhibition?

Back 20

Because there is so much substrate, it is constantly reacting with the enzyme and the inhibitor doesn't have the chance to bind to the its active site.

Front 21

How can proteins be covalently modified?

Back 21

A. Breaking peptide bonds
B. R-groups can be covalently modified by small molecules

Front 22

Phosphorylation chemistry, and sites of modification

Back 22

Chemistry: R-OH + HPO4 (-2) -->
R-O-PO3(-2) + H2O
Residues with hydroxyl groups (Ser, Thr, Tyr)

Front 23

N-Glycosylation chemistry and sites of modification

Back 23

Chemistry: R-NH-Sugar

Common site of modification: Asn

Front 24

O-Glycosylation chemistry and sites of modifications

Back 24

Chemistry: R-O-Sugar

Common site of modification: Ser, Thr, and modified residues

Front 25

Hydroxylation chemistry and sites of modifications

Back 25

Chemistry: hydroxyl group (OH) added to R groups

Common site of modification: Pro, Lys

Front 26

Carboxylation chemistry and common sites of modification

Back 26

Chemistry: carboxyl group (COOH) added to R group

Common sites of attachment: Glu

Front 27

Allosteric Inhibitors

Back 27

Bind to the allosteric site on an enzyme that when bound stablilizes the enzyme in its T-state

Front 28

General Observations about allosterically-inhibited (and activated) enzymes

Back 28

A. Almost always 2+ subunits, (regulatory and catalytic subunits)
B. Enzyme does not follow Michaelis-Menten behavior, curve is S-shaped
C. the inhibitor tends to stabilize the enzyme in T-state
D. Allosteric activators push the velocity curve more towards normal Michaelis-Menten behavior

Front 29

Tense state, and Relaxed state of allosteric enzyme

Back 29

T-state binds the substrate poorly and is a poor enzyme

R-state binds the substrate well, and is enzymatically active

Front 30

Equilibrium constant for reaction:
A+B <--> C + D

Back 30

Keq=([C][D])/([A][B])

Front 31

What is chemist's standard state?

Back 31

Chemist's standard state
1M, 1atm, 25ºC

Front 32

What is the biochemist's standard state?

Back 32

Biochemist's standard state
1M, 1atm, 25ºC, ph=7

Front 33

How is Free Energy (in biochemist's standard) defined in equilibrium terms?

Back 33

ΔGº'=-R*T*ln(Keq)

Front 34

How is Free Energy defined so that it is calculatable at any temperature, pressure and concentration?

Back 34

ΔG=ΔGº' + R*T*ln([C][D]/[A][B])

Front 35

Define Enthalpy

Back 35

ΔH, is a measure in the difference in heat between the products and substrates.

Front 36

Define Entropy

Back 36

ΔS, is a measure of the difference in randomness or disorder between the products and substrates

Front 37

How are enthalpy and entropy related to free energy?

Back 37

ΔG=ΔH-TΔS

Front 38

In which direction will a reaction proceed if ΔG<0?

Back 38

The reaction proceeds spontaneously toward product formation

Front 39

In which direction will a reaction proceed if ΔG=0?

Back 39

No where, the reaction is in equilibrium.

[C][D]=[A][B]

Front 40

Simple definition of high-energy bonds

Back 40

Any bonds with ΔGº' less than or equal to -5 kcal/mol

Front 41

Levinthal paradox (protein folding problem)

Back 41

A 100-amino acid protein where each residue could assume any of 3 different positions (of only one of which was correct), and if it only took 10^-13 secs to convert one structure to the another, it would take over 1.6*10^27 years of random searching to acquire the native state.

Front 42

Protein folding problem put simply

Back 42

What information is contained in the primary structure of a protein that aids its formation of tertiary structure?

Front 43

In what state is a protein the most stable?

Back 43

Its native state, where it tends to have its lowest energy state.

Front 44

The free energy of folding of proteins...

Back 44

tends to be negative, because native state forms spontaneously, but the free energy isn't too high (5-10 kcal/mol) because proteins need to remain dynamic

Front 45

Two primary forces that drive formation of native state of protein

Back 45

Hydrophobic effect
Hydrogen bonding

Front 46

Forces that favor formation of unfolded protein

Back 46

1. Conformational entropy (flipside of hydrophobic effect)
2. Enthalpic cost of burying polar groups that are not in hydrogen bonds

Front 47

How is ΔGfold for a protein determined

Back 47

Free energy that favors native state MINUS free energy that favors unfolded state

Front 48

Describe a Ramachandran plot

Back 48

A plot of frequently observed phi versus psi angles

Front 49

Phi and psi angles

Back 49

Two bond angles around the alpha C in a polypeptide, only bond angles around the alpha C that are allowed to rotate

Front 50

Chaperones

Back 50

Enzymes that assist in protein folding

Front 51

Two major classes of chaperones

Back 51

Hsp70 and chaperonins

Front 52

Protein disulfide isomerase

Back 52

Ensures that disulfide bonds are properly formed

Front 53

Peptide prolyl cis-trans isomerase

Back 53

Catalyzes formation of the most stable configuration of proline residues

Front 54

Ways that lead to denatured proteins...

Back 54

1. A tiny percent of cells are unfolded from native state at any given time.
2. Mutant version of the protein favors unfolded state
3. Have two low-energy stable states, examples include prion diseases or tranmissible spongiform encephalopathies.

Front 55

Motor proteins convert ____ energy into ____ forces.

Back 55

Chemical energy into physical forces

Front 56

Ways physical forces are used in cells

Back 56

1. Moving physical structures, like organelles and chromosomes
2. remolding the shapes of molecules
3. Muscle contraction

Front 57

What makes up the light band in a skeletal muscle cell?

Back 57

Thin filaments, or actin

Front 58

What makes up the dark band in a skeletal muscle cell?

Back 58

Thick filaments, or myosin

Front 59

How do actin filaments form?

Back 59

Actin monomers aggregate together, forming a filament that has two distinct ends (polar).

Front 60

How is myosin formed?

Back 60

Myosin dimers, form a coiled-coil (intertwined alpha-helices), capped off by a head region and neck region. Coiled-coil regions wrap together, to form the thick filament

Front 61

In resting state, the myosin head is...

Back 61

bound to a monomer of the actin filament.

Front 62

What happens when the myosin head binds ATP?

Back 62

The myosin head releases the actin filament.

Front 63

What happens when the ATP bound to the myosin head is hydrolyzed?

Back 63

A conformational change occurs in the head domain, and a less stable state is formed.

Front 64

What allows the myosin head to bind to an actin monomer again?

Back 64

The release of Pi

Front 65

In which direction does the myosin head move when binding actin monomers?

Back 65

Once Pi is released, the head region is in a different confirmation so it binds the next actin monomer in the "plus" end direction.

Front 66

What happens when ADP is released from the myosin head?

Back 66

The apo-myosin head group spontaneously forms its most stable state, without releasing actin. This is the POWER STROKE.

Front 67

What is the power stroke?

Back 67

The impetus behind the sliding of the actin and myosin filaments in muscle contraction.

Front 68

How are muscle cells regulated to contract?

Back 68

The central nervous system modulates the intracellular [Ca++].

Front 69

What is the intracellular [Ca++] in a resting cell?

Back 69

Low concentration, <1 micromolar

Front 70

Aspartate transcarbamylase (ATCase) is subject to what type of regulation?

Back 70

Allosteric regulation

Front 71

What happens to K 0.5, for ATCase when ATP is added to solution?

Back 71

K 0.5, is reduced. ATP is an activator for ATCase and stabilizes the R state.

Front 72

What happens to K 0.5, for ATCase when CTP is added to solution?

Back 72

K 0.5 increases. CTP acts as an inhibitor for ATCase, because it stabilizes the T state.

Front 73

What happens to Vmax for ATCase when CTP and ATP are added to solution?

Back 73

Nothing.

Front 74

What reaction does ATCase catalyze?

Back 74

The pathway by which CTP (cytidine triphosphate) is produced.

Front 75

Tropomyosin, Troponin C (and T & I)

Back 75

A protein that attaches to the actin filament and blocks the attachment sites for the myosin head groups.

Front 76

What happens around actin filaments upon intracellular Ca++ release?

Back 76

The tropomyosin/troponin complex that was bound to the actin filament releases, because Ca++ binds to Troponin C causing a confirmational change.

Front 77

Other molecular motors

Back 77

1. kinesins, which carry out transport of cellular organelles.
2. helicases, use energy from NTP hydrolysis cycle to unwind duplex nucleic acids.

Front 78

HemoglobinA (HbA) structure

Back 78

Composed of four polypeptide chains, two alpha chains and two beta chains.

Front 79

Tertiary structure of alpha and beta globins

Back 79

Globin folds

Front 80

Heme group

Back 80

Noncovalently bound prosthetic group found in globins in HbA.
Combination of protoporphyrin IX and Fe(II).

Front 81

How is hemoglobin able to serve as an oxygen carrier?

Back 81

Oxygen binds to the Fe(II) of the heme,and the nonpolar environment of the surrounding globin chain makes oxidation of the iron Fe(III) reversible upon dissociation of the bound oxygen.

Front 82

What are the partial pressures of oxygen in the alveoli of the lungs, the arterial blood and the capillary bed?

Back 82

100 torr in alveoli
95 torr in arterial blood
20-40 torr in capillary bed

Front 83

Myoglobin

Back 83

A single globin chain that has a similar tertiary structure to that of globins in HbA.

Found in heart and skeletal muscle where it serves as an oxygen reserve.

Front 84

What happens to the affinity of hemoglobin to O2 as O2 is bound?

Back 84

The affinity increases, because it has low affinity before the first O2 is bound, but upon binding of the first O2 there is stabilization of the structure resulting in a higher affinity for O2.

Front 85

What does 2,3 Bisphosphoglycerate do?

Back 85

It lowers the affinity of hemoglobin for oxygen, by binding to the central cavity of deoxyHb (but not to the fully oxygenated form) and thus favors oxygen release.

Front 86

What is the difference in HbF to HbA and, why is that important?

Back 86

HbF is comprised of two alpha glofin chaing and 2 gamma chains. This reduces its affinity towards 2,3-BPG. This decreased affinity allows for the transfer of oxygen from maternal blood to fetal blood.

Front 87

What conditions alter amounts of 2,3 BPG present in RBCs, and in which direction do they alter it?

Back 87

Hypoxemia conditions, such as low cardiac output, lung disease, anemias, low pO2 (high altitudes), and sickle cell anemias. These conditions increase [2,3 BPG]

Front 88

What is the Bohr effect?

Back 88

The affects of [H+] on oxygen binding.

Front 89

What will decreased pH do to the affinity of oxygen to Hb?

Back 89

It will reduce that affinity, thus promoting unloading of oxygen.

Front 90

What will a low PCO2 do to hemoglobin and oxygen?

Back 90

It will elevate pH, thereby increasing oxygen affinity of O2 to Hb.

Front 91

What is the oxygen binding equilibria of Hemoglobin?

Back 91

Hb + 4O2 <--> Hb(O2)4 + 2.4H+

Front 92

What else binds to hemoglobin?
(besides, O2, and 2,3 BPG)

Back 92

1. CO2, when bound it decreases Hb oxygen affinity by an allosteric mechanism.
2. Chloride ions, also when bound decrease Hb oxygen affinity.

Front 93

What else can affect hemoglobin binding?

Back 93

Temperature.
Increased temp weakens oxygen binding.

Front 94

What is the pH around and in the RBC?

Back 94

Internal pH of the RBC is normally .1-.2 pH units lower than the plasma pH.

Front 95

What affect does pH in the plasma and RBC have on oxygen transport?

Back 95

A lower pH in the RBC promotes oxygen transport.

Front 96

How is the majority of CO2 transported in the blood?

Back 96

1. As part of the H2CO3 <--> H+ + HCO3- buffer system

2. Bound to the four alpha amino groups of Hb

Front 97

Blood buffer systems

Back 97

RBC phosphates and plasma protein

Front 98

CO2 transport in the blood produces what type of change in pH?

Back 98

None. It is called "isohydric", a product of the linked Hb-O2-CO2 reactions.

Front 99

Antibody structure

Back 99

A pair of identical light chain proteins, and a pair of identical heavy chaing proteins.

Front 100

5 major classes of antibodies

Back 100

IgM, IgD, IgG, IgA, and IgE

Front 101

What is the most abundant immunoglobin in human blood?

Back 101

IgG, which comprises ~75% of total immunoglobin content

Front 102

Briefly define the role of antibodies.

Back 102

Major role is to bind to foreing molecules, called antigens, and to then initiate an immune response in defense of the organism from pathogens.

Front 103

Where do antigens bind on the antibody?

Back 103

They bind to the N-terminal end, in a site that is formed by the light and heavy chain elements.

Front 104

Polyclonal antibody

Back 104

Heterogeneous collection of many IgGs, each of which recognizes a different sequence of a particular antigen.

Front 105

Monoclonal antibody

Back 105

Homogeneous antibody that only recognizes one epitope (sequence recognized by antibody) on the antigen.

Front 106

Immunoglobulin domains in general

Back 106

Compact domain of antibody formed by heavy and light chains.
Has two adjacent layers of antiparallel beta-sheets.

Front 107

Immunoglobulin domains of heavy chain

Back 107

VH, CH1, CH2, CH3
Variable domain, and constant domains 1, 2 and 3. (H subscript indicates they are on heavy chain)

Front 108

Immunoglobulin domains of light chain

Back 108

VL, CL
Variable domain and constant domain

Front 109

Variable domain structure on antibody

Back 109

Contains three hypervariable loops, which serve as antigen-binding site

Front 110

Group 1 collagen

Back 110

Types I, II, III and V
Molecules composed of long polypeptide chains wound into a single helical domain

Front 111

Group 2 collagens

Back 111

Types IV, VI, VII and VIII
Composed of long chains arranged in several helical segments separated by nonhelical segments

Front 112

Group 3 collagens

Back 112

Comprised of shorter chains, arranged in 1 or more helical segments.

Front 113

Two unusual features of topocollagen molecules

Back 113

1. High-content of glycine, ~ 1/3 of residues. Very small R-group allows for turning

2. High content of proline, and 4-Hydroxyproline. Because of the "locked" rotation.

Front 114

Vitamin C role in collagen formation

Back 114

ascorbic acid appears to hydrolyze proline, an important step in collagen formation

Front 115

Three mechanisms of stopping blood flow

Back 115

1. Vasoconstriction in area of trauma
2. Platelets clump and mechanically obstruct the flow of blood.
3. Insoluble, cross-linked masses of protein and trapped caells are deposited by the clotting process.

Front 116

Mechanism of vasoconstriction

Back 116

Platelets disintegrate when in contact with foreign surface, especially collagen. Clumping of platelets causes them to release serotonin - a vasoconstrictor.

Front 117

Aggregation of platelets is stimulated by...

Back 117

ADP (adenosine diphosphate), which is released by clumped platelets at site of trauma.

Front 118

Hydrolysis reaction

Back 118

A molecule of water is added across the peptide bond, to give back the amino and carboxyl groups.

Front 119

Peptidases and proteases are ___ enzymes

Back 119

Proteolytic enzymes that catalyze the hydrolysis of peptide bonds.

Front 120

Inactive clotting factor precursors

Back 120

Zymogens

Front 121

Inactive precursor of thrombin

Back 121

Prothrombin

Front 122

Thrombin cleaves what peptide?

Back 122

Cleaves fibrinogen into fibrin and frinopeptides A and B

Front 123

Fibrinogen structure

Back 123

three pairs of nonidentical polypeptides called the alpha, beta and gamma chains.

Front 124

Fibrin stabilizing factor (XIIIa)

Back 124

Specifically crosslinks the fibrin molecules

A transglutaminase, catalyzing the transfer of the R-group carbonyl of a glutamine residue to an amine group of a lysine residue on another chain.

Front 125

Fibrin stabilizing factor (XIIIa) is formed in what reaction?

Back 125

Reaction of thrombin and Ca++ with inactive zymogen, factor XIII

Thrombin cleaves a specific bond in each of the "a" subunits, releasing two peptides. Second reaction, requires Ca++ ions, b subunits are lost to give the full active enzyme.

Front 126

Precursor of prothrombin

Back 126

Preprothrombin

Front 127

How does factor Va accelerate the activation of prothrombin to thrombin?

Back 127

1. Increases local enzyme (Xa) and substrate (prothrombin) concentrations.
2. Orienting the enzyme and/or substrate molecules.
3. Increasing the intrinsic catalytic activity (Vmax) of Xa.
4. Making the substrate bonds more accessible or labile by changing the conformation of prothrombin.

Front 128

Modifications of preprothrombin after synthesis

Back 128

1. Carboxylation of some glutamic acid residues near the N-terminal end of the molecule
2. Then, resulting dicarboxylic R-groups form a calcium ion binding site.
3. Negatively charged phospholipid binds to calcium.
4. Phospholipid then binds to prothrombin via gamma carboxyglutamic acid residues.
5. Proteolytic cleavage by factor Xa.

Front 129

Proteolytic cleavage of prothrombin by what factor reults in activation?

Back 129

Factor Xa cleaves of an N-terminal peptide and then an internal bond within a disulfide loop to give a two chain structure.

Front 130

Role of vitamin K and CO2 in activation of thrombin

Back 130

Needed in order for certain glutamic acid residues on preprothrombin to be carboxylzed

Front 131

How does warfarin (or dicoumarol) work?

Back 131

Blocks the formation of Vitamin K epoxide into Vitamin K-H2, and because Vitamin K is needed for the activation of Factor X and thrombin, and it is only in the liver in limited amounts this greatly reduces the formation of blood clots.

Front 132

What are the structures of warfarin and dicoumarol?

Back 132

They are structural analogs of Vitamin K epoxide, so they block the activity of epoxide reductase.

Front 133

Thrombin formation can be regulated in what ways?

Back 133

1. Small amounts of thrombin that are formed catalyze the activation of V to Va, increasing the rates of prothrombin activation.
2. Thrombin increases the amount of Xa present.
3. Large amounts of thrombin inactivate Va, VIIIa and Xa, limiting the reaction. It also cleaves the calcium ion binding site off of prothrombin, decreasing its rate of activation.

Front 134

Regulation of factor Xa formation by intrinsic pathway

Back 134

1. Regulatory protein factor VIII is activated by the first amounts of thrombin and Xa formed, and then is inactivated by thrombin to contain the reaction.
2. Factor Xa may activate factor IX and thus increase the amount of the enzyme required for its own formation.

Front 135

Regulation of extrinsic activation of factor Xa

Back 135

1. Factor Xa can activate factor VII, the enzyme that cleaves X to give Xa.
2. Thrombin can activate factor VII to increase the rate of Xa formation.

Front 136

Prevention of Blood Clotting

Back 136

1. Protection of the integrity of platelets.
2. Removal of calcium ions (only useful in blood outside of body)
3. Heparin
4. Vitamin K antimetabolites (warfarin, Dicoumarol)
5. Protein C, Protein S, Thrombin-Thrombomodulin System

Front 137

Removal of Calcium Ions from Blood

Back 137

Ca++ is needed for prothrombin activation and earlier reactions. So removal of Ca++ would prevent blood clotting.

A blood calcium level low enough to prevent clotting is lower than the level that would kill someone.

Front 138

Heparin mechanism

Back 138

Heparin = A sulfated carbohydrate mucopolysaccharide made in mast cells.

In presence of heparin, antithrombin III, antagonizes action of thrombin, factors IXa, Xa, XIa and XIIa.

Suggested heparin in some way "catalyzes" formation of a complex between active thrombin and antithrombin III.

Front 139

Mechanism of Vitamin K Antimetabolites

Back 139

They interfere with the proper metabolism of Vitamin K, and the carboxylation of the Vitamin K dependent factors in the liver. Secretion of prothrombin, VII, IX an X is decreased.

Front 140

Protein C, Protein S, and Thrombin-Thrombomodulin System

Back 140

Thrombin forms a complex with thrombomodulin on the surface of vascular endothelium. Then the specificity of thrombin is altered so that it activates protein C, much faster than it does free thrombin. So, actions of thrombin on fibrinogen and factor V are inhibited in the T-T complex.

Active protein C binds to protein S. This complex inactivates factors Va and VIIIa by proteolysis.

Front 141

Plasminogen

Back 141

The inactive precursor of Plasmin, which is activated by Tissue-type Plasminogen Activator (TPA).

Can also bind fibrin.

Front 142

How are clots dissolved?

Back 142

The enzyme, Plasmin (which is formed when TPA activates Plasminogen), can split fibrin and dissolve fibrin clots.

Front 143

Tissue-type Plasminogen Activator

Back 143

TPA, activates Plasmin.

Front 144

Serine Proteases involved in blood clotting

Back 144

Thrombin, Xa, IXa, XIa, XIIa

Front 145

Serine Protease Inhibitor that prevents blood clotting

Back 145

Antithrombin III, it inactivates the serine proteases: thrombin, Xa, IXa, XIa, XIIa

Front 146

Vitamin K dependent factors involved in blood clotting

Back 146

Prothrombin, X, IX, VII and Protein C and Protein S

Front 147

lysyl residue + alpha-ketoglutarate + O2 --(Fe++, Vit C, ?)--> ?

Back 147

Products: 5-hydroxy lysyl + succinate + CO2
?: lysyl 5-monooxygenase

Front 148

prolyl residue + alpha-ketoglutarate + O2 --(Fe++, Vit C, ?)-->

Back 148

Products: 4-hydroxyl prolyl residue + CO2 + succinate
?: Prolyl 4-monooxygenase

Front 149

Monooxygenases always require what?

Back 149

They require a second reducing agent, in the case we studied alpha-ketogultarate.

Front 150

Glycosylation of tropocollagen

Back 150

Occurs before secretion of procollagen from the cell. This takes place in the rough ER.