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81 Cards in this Set

  • Front
  • Back
RNAse protein class?
All-Beta strands
Which residue functions as an acid and which residue functions as a base in serine proteases?
Acid: Serine
Base: Histidie
What metal ion does Carbonic Anhydrase use?
Zn 2+

complexed by 3 imidazole rings from Histidine
What is the quaternary structure of Hemoglobin?
4 x myoglobin-like chains
all alpha helices
D2 symmetry
Which residues carry out concerted acid-base catalysis in RNAse A?
2 Histidines

His 12 = base, steals H+ from ribose's 2' hydroxyl

His 119 = acid, donates H+ to leaving ribose
Define Km, the Michaelis-Menten Constant.
Km is the substrate concentration at which Vo = 1/2 of Vmax.
What happens to the Lineweaver-Burke plot in competitive inhibition?
Slope changes.

Km appears to increase (competition for active site slows rate at which protein reaches 50% saturation)

Vmax is same because catalytic ability is the same.
What happens to the Lineweaver-Burke plot in UNcompetitive inhibition?
Y-intercept changes: Vmax decreases because catalytic ability is reduced.

Km appears to decrease because Vmax decreases as well.
What is the function of Protein Disulfide Isomerase?
Repairs/catalyzes formation of disulfide bonds in peptide chains.
What is the single greatest contributor to protein stability?
The hydrophobic effect: entropic gain of water/solvent molecules. Protein folding hides hydrophobic residues inside globular protein.
What happens to the dG (Free Energy) of a protein as it nears its ideally folded conformation?
It decreases.

quaternary structure?
what are structures of subunits?
Triple right handed a-helix
Repeating residues: Gly-Pro-Hyp
Subunits: 3 left-handed helices wound around each other
What holds collagen triple helix together?
Glycine N-H + Proline C=O hydrogen bonds between adjacent chains

How held together?
A/D residues have hydrophobic interactions where the two alpha helices meet.
What are the two amino acids with chiral side chains?
Ile, Thr
What is the direction of twist in the keratin double-helix?
Which segments of a peptide come off a gel chromatography column first?
Larger molecules, which cannot fit through the holes in the gel beads and pass through with the eluent.
What is the basis of the Edman Degradation?
It cleaves the N-terminal end of a polypeptide chain by reacting with phenyl isothiocyanate to create PTH derivative that is assayable once the peptide is subjected to acid hydrolysis.
What is the basis of Affinity Chromatography?
Column is packed with protein-specific ligand; impurities do not bind ligand and are flushed out. Protein of interest can then be cleaved from ligand to isolate.
What is the basis of protein dialysis?
Smaller proteins diffuse out through size-specific holes in the dialysis membrane; larger ones stay behind in the bag.
What is the basis of ion exchange chromatography?
Ion exchange chromatography separates proteins on the basis of how well they bind to a charged substrate at a given pH and salt concentration. Ex. if a protein has an esp. high net negative charge at pH = X, run it through a + column to separate it from a mix.
What is the basis of Sodium Dodecyl Sulfate Protein Agarose Gel Electrophoresis (SDS-PAGE)?
First SDS denatures proteins to linearize them. Then the large negative charge of SDS allows the gel to separate the pieces solely by size.

One SDS per any two residues --> charge proportional to size.

Estimate molar mass.
Where on hemoglobin does BPG bind?

What does it do?
It binds the central cavity of deoxyhemoglobin.

It reduces hg's affinity for O2 by stabilizing the deoxy-hg state.
What do areas of highly conserved protein sequence indicate?
A segment of protein that is important for proper function (i.e. active site or binding pocket)
What happens when the heme group binds Oxygen?
Fe2+ moves into the plane of the ring, pulling histidine and the a-helix with it to trigger the conformational change.
What is a sigmoidal curve indicative of?
Cooperative binding between protein subunits. Subunits must be in contact with one another and interact upon ligand binding.
What does the Bohr Effect have to do with Hemoglobin?
Ion pairs are disrupted in deoxyhemoglobin and get deprotonated in oxy state (Hb releases protons when it binds O2)

In acidic conditions these ion pair forming residues are protonated and stabilize/encourage hg to adopt deoxy state.

High H+ --> lower O2 affinity
Describe the SYMMETRY MODEL of allosterism.
Symmetry must be maintained (all or nothing change T/R states)

Ligand can bind either T or R state

Binding encourages shift to R state.
Describe the SEQUENTIAL MODEL of allosterism.
Binding of subunit necessitates conformational change.

Symmetry need not be maintained

Binding in one increases binding in neighbors through slight structural changes.
What is the protein class of calmodulin?

Two domains

Undergoes conformational change upon ligand binding.
What is the critical flaw in sickle cell hemoglobin, HbS?
Glu6 --> Val6

Val fits in a hydrophobic pocket in other HbS or Hb and makes them stick together.
What is the best buffer range for weak acids and bases?
pK +/- 1 pH point
What is the definition of Kcat?
The ratio of product produced per unit of enzyme. Kcat = K2 ish.

A measure of enzyme's efficiency.
What is alpha in the context of enzyme kinetics?
in competitive inhibition, it is the factor by which [S] must increase to overcome impact of [I]
What is the mechanism of neuraminidase?
Neuraminidase = influenza protein.

Cleaves sialic acid from the membrane of host cell to allow new virus particles to escape.
What is a Michaelis Complex?
ES, the enzyme/bound substrate complex.
What is the x-intercept on the Lineweaver-Burke Plot?
What is the y-intercept on the Lineweaver-Burke Plot?
Define "prochiral."
An achiral molecule that can be made chiral in one step (one switch, etc).

Ex. Citrate --> Isocitrate
What is the structure and class of neuraminidase?
4 domains of all wiggly beta strands
What are the final two proteins involved in the blood coagulation cascade?
Prothrombin --> Thrombin


Fibrinogen --> Fibrin
What are two ways the body regulates enzyme activity?
1. make inhibitors

2. make proenzymes and activate as necessary.
What makes DANA a suitable inhibitor?
It is a neuraminidase transition state analog

Planar like transition state of sialic acid

4-guanidino group makes ion pair with glutamates in active site --> high affinity for neuraminidase.
Is it better for an enzyme to bind the substrate or the transition state tightly?
Transition state. Stabilizing the transition state lowers the activation energy of the reaction.
In the serine proteases, which residue acts as a base and which acts as an acid?
His is a base taking H+ from Ser (acid)

His is an acid donating H+ to leaving peptide

His is a base abstracting H+ from H2O, then an acid donating to Ser (base).
What is a prosthetic group?
An organic or inorganic coenzyme that is covalently attached to the enzyme.
What is the Reaction Coordinate?
The reaction path of lowest free energy.
Why is the peptide bond planar?
Resonance between the N lone pair and the carbonyl double bond gives the C-N peptide bond partial double bond character.
Which side chains are aliphatic?
Gly, Ala, Val, Ile, Leu, Pro
Which side chains are aromatic?
Phe, Tyr, Trp
Which side chains are polar and uncharged?
Met, Ser, Thr, Cys, Asn, Gln
Which side chains are negatively charged (acidic)?
Asp, Glu
Which side chains are positively charged (basic)?
Arg, Lys, His
What is the structure and class of myoglobin?
All alpha

Eight helices

Monomeric Globular unit, Soluble
What is the structure and class of ATCase?
C6R6 (Catalytic, Regulatory)

D3 symmetry

mixed a/b domains
What is actin made up of?
Repeating G-Actin subunits arranged in helical symmetry
What molecules inhibit ATCase and how?
CTP through feedback inhibition (allosteric modification)

PALA through competitive inhibition (bisubstrate analog)
What molecule activates/stimulates ATCase?
ATP through allosteric modification

ATP binds ATCase away from the active site.
What enzyme activates chymotrypsinogen?

Later pi-chymotrypsin activates itself and makes alpha-chymotrypsin.
Which three endopeptidases (of the six we studied) are serine proteases?
What is the mechanism of RNAse A?
Two Histidines at active site --> concerted acid/base catalysis to form cyclic ribose phosptate intermediate
What is the difference between a cis and trans peptide?
Orientation of side chains w/respect to peptide bond.

Cis = on same side
Trans = on opposite sides (usually more stable)
What two important biological molecules are derived from tyrosine?
What is the name for aggregates of misfolded proteins?

Alzheimer's, Transthyretin diseases, Mad Cow Disease (prions)
What are the alpha-keratin helix specs?

handedness, length, pitch, etc.
Right handed helix
450 Angstroms long
310 residues long
3.5 residues/turn
5.1 Angstrom pitch
What was significant about Adenosine and GAPDH?
Hydrophobic substituent on Adenosine fit nicely into groove on sleeping sickness parasite GAPDH enzyme.

Inhibitor drug design
Class and structure of Trypsin?
All beta protein

Monomeric unit

Serine Protease
What three methods of covalent catalysis are utilized by serine proteases?
1. concerted acid/base catalysis
2. transition state stabilization (oxyanion hole)
3. covalent catalysis
How are proteases more frequently controlled?
Synthesis of a protease inhibitor molecule (ex. PTI, Pancreatic Trypsin Inhibitor)
What family of enzymes does Thrombin belong to?
Serine Proteases
Which conformation of ATCase is favored by binding of CTP?
Closed "T" conformation, less active.
PALA binds the active sites of ATCase, causing a conformational change from T --> active R.

Why does it still inhibit catalysis?
It doesn't let go of the active site. So even though ATCase is in active state it can't catalyze reactions.
How many molecules of sialic acid can neuraminidase bind?
Four: one per wiggly all-beta subunit.
What is the symmetry of GroES?
What is the symmetry of GroEL?
Where do side chains point in a beta sheet?
Alternating above/below the plane of the sheet.
Where do side chains point in an alpha helix?
Toward the outside of the helix.
Where are active sites most frequently found?
At the junctions between adjacent protein domains.
How much more stable is folded vs unfolded protein?
dG = ~0.4 kJ/residue/mol.

100-residue protein: only ~40kJ/mol more stable
Describe the three-step renaturation process.
1. Expose to oxygen to reoxidize (reform) scrambled disulfide bonds.
2. Remove Urea to allow folding. (still scrambled)
3. Add catalytic amount of beta-mercapthoethanol to repair scrambled disulfide bonds

--> renatured protein.
What is the significance of the Hill Coefficient?
Steepness of Hemoglobin Binding Curve

Steeper Hill Coefficient --> faster change in Hg O2 saturation/unloading.