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150 Cards in this Set

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Competitive Inhibition - Lineweaver Burke Equation
1/v=(1+[I/Ki])*(Km/Vmax)*(1/[S]) + 1/Vmax
Non-Competitive Inhibition - Lineweaver Burke Equation
1/v=(1+[I/Ki])*(Km/Vmax)*(1/[S]) + (1+[I/Ki])*1/Vmax
Uncompetitive Inhibition - Lineweaver Burke Equation
1/v=(Km/Vmax)*(1/[S]) + (1+[I/Ki])*1/Vmax
No inhibitor - Lineweaver Burke Equation
1/v=Km/Vmax)*(1/[S]) + 1/Vmax
No inhibitor - Michaelis Menten Equation
pK for alpha-carboxyl group (free)
pK for beta-carboxyl group
pK for imidazole
pK for sulfhydryl
pK for primary alpha-amino (free)
pK for epsilon amino, or phenolic hydroxyl
pK for secondary alpha amino (free)
pK for guanido
Amino acids with carboxyl groups
Glutamate, Aspartate
Amino acids with aromatic side groups
Tryptophan and Phenylalanine
What happens to a R group at pH above it's pk?
R group will be less protonated
What happens to an R group at a pH equal to it's pK?
50% of the R groups will be protonated, and 50% will be unprotonated
What happens to an R group at a pH below its pK?
R group will be more protonated
Enzymes alter the ____ energy of a reaction, no its change in ____ energy.
They alter the ACTIVATION energy, and do not change FREE energy.
How does high substrate concentration affect irreversible inhibitors/inhibition?
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.
How can proteins be covalently modified?
A. Breaking peptide bonds
B. R-groups can be covalently modified by small molecules
Phosphorylation chemistry, and sites of modification
Chemistry: R-OH + HPO4 (-2) -->
R-O-PO3(-2) + H2O
Residues with hydroxyl groups (Ser, Thr, Tyr)
N-Glycosylation chemistry and sites of modification
Chemistry: R-NH-Sugar

Common site of modification: Asn
O-Glycosylation chemistry and sites of modifications
Chemistry: R-O-Sugar

Common site of modification: Ser, Thr, and modified residues
Hydroxylation chemistry and sites of modifications
Chemistry: hydroxyl group (OH) added to R groups

Common site of modification: Pro, Lys
Carboxylation chemistry and common sites of modification
Chemistry: carboxyl group (COOH) added to R group

Common sites of attachment: Glu
Allosteric Inhibitors
Bind to the allosteric site on an enzyme that when bound stablilizes the enzyme in its T-state
General Observations about allosterically-inhibited (and activated) enzymes
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
Tense state, and Relaxed state of allosteric enzyme
T-state binds the substrate poorly and is a poor enzyme

R-state binds the substrate well, and is enzymatically active
Equilibrium constant for reaction:
A+B <--> C + D
What is chemist's standard state?
Chemist's standard state
1M, 1atm, 25ºC
What is the biochemist's standard state?
Biochemist's standard state
1M, 1atm, 25ºC, ph=7
How is Free Energy (in biochemist's standard) defined in equilibrium terms?
How is Free Energy defined so that it is calculatable at any temperature, pressure and concentration?
ΔG=ΔGº' + R*T*ln([C][D]/[A][B])
Define Enthalpy
ΔH, is a measure in the difference in heat between the products and substrates.
Define Entropy
ΔS, is a measure of the difference in randomness or disorder between the products and substrates
How are enthalpy and entropy related to free energy?
In which direction will a reaction proceed if ΔG<0?
The reaction proceeds spontaneously toward product formation
In which direction will a reaction proceed if ΔG=0?
No where, the reaction is in equilibrium.

Simple definition of high-energy bonds
Any bonds with ΔGº' less than or equal to -5 kcal/mol
Levinthal paradox (protein folding problem)
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.
Protein folding problem put simply
What information is contained in the primary structure of a protein that aids its formation of tertiary structure?
In what state is a protein the most stable?
Its native state, where it tends to have its lowest energy state.
The free energy of folding of proteins...
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
Two primary forces that drive formation of native state of protein
Hydrophobic effect
Hydrogen bonding
Forces that favor formation of unfolded protein
1. Conformational entropy (flipside of hydrophobic effect)
2. Enthalpic cost of burying polar groups that are not in hydrogen bonds
How is ΔGfold for a protein determined
Free energy that favors native state MINUS free energy that favors unfolded state
Describe a Ramachandran plot
A plot of frequently observed phi versus psi angles
Phi and psi angles
Two bond angles around the alpha C in a polypeptide, only bond angles around the alpha C that are allowed to rotate
Enzymes that assist in protein folding
Two major classes of chaperones
Hsp70 and chaperonins
Protein disulfide isomerase
Ensures that disulfide bonds are properly formed
Peptide prolyl cis-trans isomerase
Catalyzes formation of the most stable configuration of proline residues
Ways that lead to denatured proteins...
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.
Motor proteins convert ____ energy into ____ forces.
Chemical energy into physical forces
Ways physical forces are used in cells
1. Moving physical structures, like organelles and chromosomes
2. remolding the shapes of molecules
3. Muscle contraction
What makes up the light band in a skeletal muscle cell?
Thin filaments, or actin
What makes up the dark band in a skeletal muscle cell?
Thick filaments, or myosin
How do actin filaments form?
Actin monomers aggregate together, forming a filament that has two distinct ends (polar).
How is myosin formed?
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
In resting state, the myosin head is...
bound to a monomer of the actin filament.
What happens when the myosin head binds ATP?
The myosin head releases the actin filament.
What happens when the ATP bound to the myosin head is hydrolyzed?
A conformational change occurs in the head domain, and a less stable state is formed.
What allows the myosin head to bind to an actin monomer again?
The release of Pi
In which direction does the myosin head move when binding actin monomers?
Once Pi is released, the head region is in a different confirmation so it binds the next actin monomer in the "plus" end direction.
What happens when ADP is released from the myosin head?
The apo-myosin head group spontaneously forms its most stable state, without releasing actin. This is the POWER STROKE.
What is the power stroke?
The impetus behind the sliding of the actin and myosin filaments in muscle contraction.
How are muscle cells regulated to contract?
The central nervous system modulates the intracellular [Ca++].
What is the intracellular [Ca++] in a resting cell?
Low concentration, <1 micromolar
Aspartate transcarbamylase (ATCase) is subject to what type of regulation?
Allosteric regulation
What happens to K 0.5, for ATCase when ATP is added to solution?
K 0.5, is reduced. ATP is an activator for ATCase and stabilizes the R state.
What happens to K 0.5, for ATCase when CTP is added to solution?
K 0.5 increases. CTP acts as an inhibitor for ATCase, because it stabilizes the T state.
What happens to Vmax for ATCase when CTP and ATP are added to solution?
What reaction does ATCase catalyze?
The pathway by which CTP (cytidine triphosphate) is produced.
Tropomyosin, Troponin C (and T & I)
A protein that attaches to the actin filament and blocks the attachment sites for the myosin head groups.
What happens around actin filaments upon intracellular Ca++ release?
The tropomyosin/troponin complex that was bound to the actin filament releases, because Ca++ binds to Troponin C causing a confirmational change.
Other molecular motors
1. kinesins, which carry out transport of cellular organelles.
2. helicases, use energy from NTP hydrolysis cycle to unwind duplex nucleic acids.
HemoglobinA (HbA) structure
Composed of four polypeptide chains, two alpha chains and two beta chains.
Tertiary structure of alpha and beta globins
Globin folds
Heme group
Noncovalently bound prosthetic group found in globins in HbA.
Combination of protoporphyrin IX and Fe(II).
How is hemoglobin able to serve as an oxygen carrier?
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.
What are the partial pressures of oxygen in the alveoli of the lungs, the arterial blood and the capillary bed?
100 torr in alveoli
95 torr in arterial blood
20-40 torr in capillary bed
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.
What happens to the affinity of hemoglobin to O2 as O2 is bound?
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.
What does 2,3 Bisphosphoglycerate do?
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.
What is the difference in HbF to HbA and, why is that important?
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.
What conditions alter amounts of 2,3 BPG present in RBCs, and in which direction do they alter it?
Hypoxemia conditions, such as low cardiac output, lung disease, anemias, low pO2 (high altitudes), and sickle cell anemias. These conditions increase [2,3 BPG]
What is the Bohr effect?
The affects of [H+] on oxygen binding.
What will decreased pH do to the affinity of oxygen to Hb?
It will reduce that affinity, thus promoting unloading of oxygen.
What will a low PCO2 do to hemoglobin and oxygen?
It will elevate pH, thereby increasing oxygen affinity of O2 to Hb.
What is the oxygen binding equilibria of Hemoglobin?
Hb + 4O2 <--> Hb(O2)4 + 2.4H+
What else binds to hemoglobin?
(besides, O2, and 2,3 BPG)
1. CO2, when bound it decreases Hb oxygen affinity by an allosteric mechanism.
2. Chloride ions, also when bound decrease Hb oxygen affinity.
What else can affect hemoglobin binding?
Increased temp weakens oxygen binding.
What is the pH around and in the RBC?
Internal pH of the RBC is normally .1-.2 pH units lower than the plasma pH.
What affect does pH in the plasma and RBC have on oxygen transport?
A lower pH in the RBC promotes oxygen transport.
How is the majority of CO2 transported in the blood?
1. As part of the H2CO3 <--> H+ + HCO3- buffer system

2. Bound to the four alpha amino groups of Hb
Blood buffer systems
RBC phosphates and plasma protein
CO2 transport in the blood produces what type of change in pH?
None. It is called "isohydric", a product of the linked Hb-O2-CO2 reactions.
Antibody structure
A pair of identical light chain proteins, and a pair of identical heavy chaing proteins.
5 major classes of antibodies
IgM, IgD, IgG, IgA, and IgE
What is the most abundant immunoglobin in human blood?
IgG, which comprises ~75% of total immunoglobin content
Briefly define the role of antibodies.
Major role is to bind to foreing molecules, called antigens, and to then initiate an immune response in defense of the organism from pathogens.
Where do antigens bind on the antibody?
They bind to the N-terminal end, in a site that is formed by the light and heavy chain elements.
Polyclonal antibody
Heterogeneous collection of many IgGs, each of which recognizes a different sequence of a particular antigen.
Monoclonal antibody
Homogeneous antibody that only recognizes one epitope (sequence recognized by antibody) on the antigen.
Immunoglobulin domains in general
Compact domain of antibody formed by heavy and light chains.
Has two adjacent layers of antiparallel beta-sheets.
Immunoglobulin domains of heavy chain
VH, CH1, CH2, CH3
Variable domain, and constant domains 1, 2 and 3. (H subscript indicates they are on heavy chain)
Immunoglobulin domains of light chain
Variable domain and constant domain
Variable domain structure on antibody
Contains three hypervariable loops, which serve as antigen-binding site
Group 1 collagen
Types I, II, III and V
Molecules composed of long polypeptide chains wound into a single helical domain
Group 2 collagens
Types IV, VI, VII and VIII
Composed of long chains arranged in several helical segments separated by nonhelical segments
Group 3 collagens
Comprised of shorter chains, arranged in 1 or more helical segments.
Two unusual features of topocollagen molecules
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.
Vitamin C role in collagen formation
ascorbic acid appears to hydrolyze proline, an important step in collagen formation
Three mechanisms of stopping blood flow
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.
Mechanism of vasoconstriction
Platelets disintegrate when in contact with foreign surface, especially collagen. Clumping of platelets causes them to release serotonin - a vasoconstrictor.
Aggregation of platelets is stimulated by...
ADP (adenosine diphosphate), which is released by clumped platelets at site of trauma.
Hydrolysis reaction
A molecule of water is added across the peptide bond, to give back the amino and carboxyl groups.
Peptidases and proteases are ___ enzymes
Proteolytic enzymes that catalyze the hydrolysis of peptide bonds.
Inactive clotting factor precursors
Inactive precursor of thrombin
Thrombin cleaves what peptide?
Cleaves fibrinogen into fibrin and frinopeptides A and B
Fibrinogen structure
three pairs of nonidentical polypeptides called the alpha, beta and gamma chains.
Fibrin stabilizing factor (XIIIa)
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.
Fibrin stabilizing factor (XIIIa) is formed in what reaction?
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.
Precursor of prothrombin
How does factor Va accelerate the activation of prothrombin to thrombin?
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.
Modifications of preprothrombin after synthesis
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.
Proteolytic cleavage of prothrombin by what factor reults in activation?
Factor Xa cleaves of an N-terminal peptide and then an internal bond within a disulfide loop to give a two chain structure.
Role of vitamin K and CO2 in activation of thrombin
Needed in order for certain glutamic acid residues on preprothrombin to be carboxylzed
How does warfarin (or dicoumarol) work?
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.
What are the structures of warfarin and dicoumarol?
They are structural analogs of Vitamin K epoxide, so they block the activity of epoxide reductase.
Thrombin formation can be regulated in what ways?
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.
Regulation of factor Xa formation by intrinsic pathway
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.
Regulation of extrinsic activation of factor Xa
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.
Prevention of Blood Clotting
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
Removal of Calcium Ions from Blood
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.
Heparin mechanism
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.
Mechanism of Vitamin K Antimetabolites
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.
Protein C, Protein S, and Thrombin-Thrombomodulin System
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.
The inactive precursor of Plasmin, which is activated by Tissue-type Plasminogen Activator (TPA).

Can also bind fibrin.
How are clots dissolved?
The enzyme, Plasmin (which is formed when TPA activates Plasminogen), can split fibrin and dissolve fibrin clots.
Tissue-type Plasminogen Activator
TPA, activates Plasmin.
Serine Proteases involved in blood clotting
Thrombin, Xa, IXa, XIa, XIIa
Serine Protease Inhibitor that prevents blood clotting
Antithrombin III, it inactivates the serine proteases: thrombin, Xa, IXa, XIa, XIIa
Vitamin K dependent factors involved in blood clotting
Prothrombin, X, IX, VII and Protein C and Protein S
lysyl residue + alpha-ketoglutarate + O2 --(Fe++, Vit C, ?)--> ?
Products: 5-hydroxy lysyl + succinate + CO2
?: lysyl 5-monooxygenase
prolyl residue + alpha-ketoglutarate + O2 --(Fe++, Vit C, ?)-->
Products: 4-hydroxyl prolyl residue + CO2 + succinate
?: Prolyl 4-monooxygenase
Monooxygenases always require what?
They require a second reducing agent, in the case we studied alpha-ketogultarate.
Glycosylation of tropocollagen
Occurs before secretion of procollagen from the cell. This takes place in the rough ER.