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394 Cards in this Set
- Front
- Back
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What are the 3 major concepts, and the corresponding scientist, that lead to the mathematical model for steady-state kinetics?
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1. Adrian Brown: ES intermediate: saturation of E when [S] is high
2. Michaelis & Menten: Equilbrium (k₁ >> k₂) 3. Briggs & Haldane: Steady-state approximation: formation of ES = disappearance of ES |
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Vmax
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-Max rate of turnover
-Unit: μM•s^-1 |
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Kcat
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-Turnover number
-Half of Vmax -Unit: s^-1 |
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What does an inhibitor do? Three things...
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1-Binds an enzyme or enzyme intermediate
2-Interferes w/ activity of enzyme 3-Can be reversible or irreversible |
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How does an Irreversible Inhibitor work? What does it do?
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-Inhibitor binds and is attached covalently
-Enzyme is inactivated -See lost of activity as a function of time -Typically do not see an effect on Km |
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What are suicide inactivators?
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-Irreversible inhibitors
-Unreactive until bind to specific active site on enzyme |
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What are the 3 classes of reversible inhibition
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1-Competitive
2-Uncompetitve 3-Mixed (noncompetitive: pure or mixed) |
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What happens in competitive inhibition?
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-Inhibitor binds to active site, therefore it competes with substrate
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How are the substrate and inhibitor related in competitive inhibition?
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Substrate and inhibitor are structurally similiar
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How can you favor the enzyme binding substrate in competitve inhibition?
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-Increase [S]
-When [S] >> [I], minimize probability of E binding to I and reaction exhibits normal Vmax -This works b/c inhibitor binds reversibly to enzyme |
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How does comeptitive inhibition affect graphical representation? Three things...
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1-No effect on kcat or Vmax
2-Require higher [S] to reach kcat 3-Increase in Km |
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How is Km affected by competitive inhibition?
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-Increased by (1 + [I]/K₁)
-Km becomes the "apparent Km" in the presence of an inhibitor |
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What happens in uncompetitive inhibition? Three things...
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1-Inhibitor binds to ES directly (not E)
2-Inhibitor binds at site distinct from substrate active site 3-Inhibitor binding results in conformational change that distorts active site |
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How does umcompetitive inhibition affect graphical representation? Two ways...
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1-Decrease in kcat (or Vmax) that cannot be reversed by increase in [S]
2-Decrease in the apparent Km |
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How does mixed inhibition work? Three things...
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1-Inhibitor can bind E or ES
2-Inhibitor does not bind active site of E 3-Usually affects both Km and kcat and increase in [S] cannot restore activity |
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If an inhibitor is not competitive then what is it referred to as?
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Allosteric Inhibitor
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Where do allosteric inhibitors bind?
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A different site than the active site
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What do serine proteases do?
What are 7 examples of them? |
They catalyze hydrolytic cleavage of peptide bonds
Examples: 1.Chymotrypsin 2.Trypsin 3.Elastase 4.Thrombin 5.Substilisin 6.Plasmin 7.Tissue plasminogen activator |
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What are four things associated with Chymotrypsin, Trypsin, and Elastase?
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1-Digestive enzymes that catalyze peptide bond cleavage
2-Synthesized in pancrease 3-Secreted in inactive precursors (zymogens) 4-Activated in digestive tract by enteropeptidase |
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What are three things associated with the activation of Chymotrypsin, Trypsin, and Elastase?
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1-Enteropeptidase
2-Specific cleavage 3-Clips of N-terminal hexapeptide for activation |
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How are Chymotrypsin, Trypsin and Elastase similiar and different?
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Similiar: AA sequence and mechanism of catalysis
Different: Each has its distinctive substrate specificity (active site has specific substrate binding region) |
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What is Chymotrypsin's cleavage specificity for activation?
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It cleaves the peptide bond adjacent to the aromatic aa
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Chymotrypsin:
What three amino acids are required for Chymotrypsin cleavage? How is it stabilized? |
The Catalytic triad:
1.Ser195 2.His57 3.Asp102 Catalytic triad is stablized by H bonds |
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Chymotrypsin:
What happens when the polypeptide chain of Chymotrypsin folds? Three parts... |
1.Active site
2.Specific substrate pocket 3.Common region for triad |
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Chymotrypsin:
What two things does binding a substrate to Chymotrypsin cause? |
1-Conformational change
2-Stronger interaction between His57 & Asp102 |
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Chymotrypsin:
What does Ser195 attack? What happens b/c of this attack? |
Ser195 attacks carbonyl of substrate
Oxygen then becomes negatively charged |
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Chymotrypsin:
What are the 2 phases in Chymotrypsin cleavage? |
1. Acylation
2. Deacylation |
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What is Acylation?
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Peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl and the enzyme
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What is Deacylation?
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The ester linkage is hydrolyzed and the non-acylated enzyme is regenerated
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Chymotrypsin:
In step 1, what happens when the substrate binds the Chymotrypsin enzyme? Two things... |
1-Side chain of aa residue adjacent to peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme
2-Peptide bond for attack is positioned |
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Chymotrypsin:
What is the role of each aa in the triad? |
-Asp102: stablilizes His57 via H-bond
-His57: abstracts proton from Ser195 -Ser195: involved in catalysis (suprise because pKa ~ 16) |
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Chymotrypsin:
In step 2 how is the ES complex formed? Three things... |
1-S binds active of enzyme
2-Proton from Ser195 removed by His 57 3-Nucleophilic (e- rich) oxygen attacks carbonyl C of peptide |
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Chymotrypsin:
What is the orientation of S when bound to active site? Where is the Phe R group? |
-Oriented so that Chymotrypsin cleaves carbonyl side hydrophobic aa
Phe R group is in specificity pocket |
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Chymotrypsin:
What does the removal of a proton from Ser195 result in? Three things... |
1-Strongly nucleophilic alkoxide ion on Ser195
2-This ion attacks the peptide carbonyl group resulting in a short-lived negative charge on carbonyl O of S 3-Form transient acyl intermediate |
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Chymotrypsin:
How is the short-lived negative charge on carbonyl O of S stablized? |
By H-bonding in the oxyanion hole
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Chymotrypsin:
What happens when the acyl transient intermediate is formed? 7 Steps... |
1-Collapse of intermediate
2-Carbonyl C=O changed to C-O 3-H-bonding to Gly193 & Ser195 4-His57 donates H+ to peptide R1-NH to facilitate cleavage 5-His57 and Asp102 share proton 6-Break peptide bond 7-Release product 1 |
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Chymotrypsin:
What role does His57 play by donating H+? |
Acid Catalyst
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Chymotrypsin:
What occurs during the second step with the acyl-enzyme intermediate? Two things... |
1-Covalent bond formation between peptide and Ser195
2-Water enters the active site and dontates proton to His57 |
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Chymotrypsin:
How is the second tetrahedral intermediate formed (ETI2)? |
-OH from water attacks carbonyl group and forms ETI2
-Oxygen is in the oxyanion hole agains taking on a negative charge |
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Chymotrypsin:
What two amino acids stablize ETI2? |
1.Ser195
and 2.Gly193 ("transistion-state-like") |
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Chymotrypsin:
What two things occurs in deacylation to form the second product? |
1-His57 donates proton to Ser195 to form second product
2-Polypeptide has a new C-terminus |
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Chymotrypsin:
How is the free enzyme regenerated? Two parts... |
1-Product is released from active site
2-Active site returns to original conformation |
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Chymotrypsin:
What are the 5 catalytic modes in Chymotrypsin catalysis? |
1. Substrate binding and orientation (proximity effect)
2. Acid-base catalysis by His57 3. Covalent catalysis by Ser195 4. H-bonds (from Ser195 and Gly193)in oxyanion hole stabilize intermediates (transition-state) 5. Transition-state stabilization of substrate for catalysis |
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Chymotrypsin:
What does a change in Kcat for Chymotrypsin catalysis indicate? Two things... |
1-Reflects ionization of His57
2-Decline in kcat at low pH b/c His57 is protonated |
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Chymotrypsin:
When is the optimum activity for Chymotrypsin? Two things... |
When...
1-His57 is deprotonated 2-IIe16 is protonated |
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What signifies enzymes that do not obey Michaelis-Menten kinetics?
Three properties... |
1-Sigmodial behavior
2-Evidence of cooperativity or allosteric regulation 3-Characterisitc of multi-subunit enzymes |
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Instead of Km, what two variables do you use for enzymes that don't obey M-M kinetics?
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S[.5]
or K[.5] |
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What are Allosteric Enzymes?
Two things... |
1- They function through reversible, noncovalent binding of reversibly compounds called allosteric modulators or effectors
2- They have active sites and one or more allosteric binding sites |
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Describe Modulators
Two things... |
1-Modulators may be inhibitory or stimulatory
2-Modulators can be homotropic (substrate and modulator are identical) or heterotropic |
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Aspartate Transcarbamoylase -ATCase
Four Things... |
1- Allosteric enzyme
2- Large, multi-subunit 3- 6 catalytic subunits 4- 6 regulatory subunits |
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What does ATCase do?
Two things... |
1-Catalyzes the first committed step in pyrimidine biosynthesis
2-Typically, [purines]=[pyrimidines] |
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What do 'ATP and CTP' do to ATCase?
Three things... |
1- ATP up regulates ATCase
2- CTP down regulates 3- Both bind at a regulatory site seperate from active site |
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What two things are unique about carbohydrates?
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1- Unique characterisitcs structurally which define functional roles
2- Structural complexity and diversity |
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What is the biological roles of carbohydrates?
Three roles... |
1- Most abundant biomolecules on earth
2- Oxidation of carbohydrates is the main energy-yielding pathway for non-photosynthetic organisms (dietary staple) 3- Insoluble polymers serve structural and protective roles |
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What are three functions of CHO polymers?
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1- Lubricate skeletal joints
2- Participate in recognition and adhesion (extracellular matrix: ECM) 3- Participate in recognition and signaling |
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What is the structure of carbohydrates?
4 parts... |
1- Polyhdyroxyl aldehydes or ketones or substances that yield aldehydes or ketones on hydrolysis
2- Empirical formula: (CH₂O)n 3- Some contain N, P, S 4- All chiral except dihydroxyacetone |
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What are the 3 major classes of carbohydrates?
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1. Monosaccharides & Disaccharides
2. Oligosaccharides: polymers of 2-20 monomeric units 3. Polysaccharides: >20 monomeric units |
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What are monosaccharides?
Four things... |
1- Simple sugars
2- Single polyhydroxyl aldehyde or ketone unit 3- Most abundant is D-glucose 4- Classified by # of C atoms (numbering begins at carbonyl C) |
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What are the properties of monosaccharides?
Four properties... |
1- Tend to be white, colorless, crystalline solids
2- Freely soluble in water 3- Insoluble in nonpolar solvents 4- Most have a sweet taste |
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Chirality and Monosaccharides?
Is it really possible? |
All except dihydroxyacetone are chiral
-Chiral C is most distant from carbonyl C -D and L enatiomers -D is common-D: OH on right side -L: OH on left side -Have n chiral centers then have 2ⁿ stereoisomers |
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What in the world is an Epimer?
What is an example? |
Two stereoisomers that differ in configuration at one asymmetric center in a compound having 2 or more asymmetric centers
-Example: D-glucose with D-mannose and D-galactose |
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When is the cyclic or ring structure more stable for monosaccharides?
Why? |
When there are 4 or more C's
Stable because Carbonyl group forms covalent bond w/ O of hydroxy group |
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In regards to energetics, why is ring structure favored?
Three reasons... |
1- Ring form is better retained in cells
2- Prevent sugars from leaving which may be neccessary at times 3- Phosphorylation prevents exit from cell |
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How is a hemiacetal or hemiketal formed?
Two things... |
1-Aldehyde or ketone reacts with alcohol in a 1:1 ratio
2-Create a new chiral center at the carbonyl C |
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How is an acetal or ketal formed?
Another two things dammit... |
1-Substitution of a second alcohol on a hemiacetal or hemiketal
2-When the second alcohol is part of another sugar molecule, a glycosidic bond is produced |
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What is a Pyranose?
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Six-membered ring compound
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What is a Furanose?
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Five-membered ring compound
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What is an Anomer?
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Isomeric forms of monosaccharides that differ only in their configuration about the hemiacetal or hemiketal carbon atom
Yeah I knew that :-) |
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Anomeric Carbon
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Hemiacetal (or carbonyl) carbon
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How are the two cyclic forms of D-glucose formed?
Two steps... |
1- Aldehyde group at C-1 and hydroxyl group at C-5 forms a hemiacetal linkage
2- Either of two stereoisomers, α & β anomers, which differ only stereochemistry at hemiacetal C |
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α vs β form
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-α: OH points down
-β: OH points up |
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What is Mutarotation?
Three parts... |
1- process by which α & β anomers of D-glucose interconvert in aqueous soln
2- Involves breaking covalent bond 3- Two configurations interconverting |
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How are disaccharides bonded?
Three things... |
1- O-glycosidic bond
2- Hydroxyl group of one sugar w/ anomeric C of another 3- Glycosidic bonds are easily hydrolyzed by acid but resist cleavage by base |
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Why is there such diversity of disaccharides and carbohydrates in general?
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The variety of O-glycosidic linkages that can be formed
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What is the reducing end in a sugar?
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-End of a chain with a free anomeric C
-C1 is NOT involved in a glycosidic linkage so can take linear form and carbonyl O is available to be oxidized |
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What makes a sugar a non-reducing sugar?
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When the anomeric C is involved in a glycosidic bond
That sugar residue cannot take the linear form |
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What is Lactose?
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D-galactose & D-glucose
-Sugar in milk -Lactose intolerant people do not have lactase to hydrolyze β 1→4 |
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What is Sucrose?
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D-glucose & D-fructose
-Table sugar |
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WTF is Trehalose?
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D-glucose
-Major constituent of circulating fluid in insects -An energy storage compound |
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What are the two main functions of polysaccharides?
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1. Storage (starch and glycogen: essentially insoluble)
2. Structural (cellulose and chitin) |
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What two characteristics are important for storage molecules?
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1- Storing largest numbers of monomers as possible
2- Limited space |
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Would monomers (glucose) be good for storage?
WHY? |
NO, CHIPPY
-Won't work b/c of H-bonding w/ water -Each OH will H-bond with water and you'd end up carrying around all that extra water |
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Why are polymers better in storage?
Two reasons... |
1- Reduce osmotic pressure
2- Decrease number of OH groups that interact with water |
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What 4 things do polysaccharides differ in?
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1. Identity of re-occuring
monosaccharides 2. Length of chains 3. Bond linking units (α or β) 4. Degree of branching |
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What is starch?
What two types of glucose polymers does it contain? |
Storage polysaccharide
Contains: Amylose and Amylopectin |
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What is Amylose?
Two things... |
1- Long, unbranched chains of D-glucose connected by α 1-4 linkages
2- Chains vary in length and MW |
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What is Amylopectin?
Three things... |
1- Highly branched D-glucose linkages are α 1-4, but branch points are α 1-6
2- Branches occur every 24-30 aa residues 3- High MW |
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What is the purpose of branching in amylopectin?
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Branching removes the OH that would interact w/ water
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What is glycogen?
Three things... |
1- Storage Polysaccharide
2- Main storage form of animal cells 3- Similar in structure to amylopectin |
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What is the structure of glycogen?
Two things... |
1- Highly branched D-glucose linkages are α 1-4, but branch points α 1-6
2- Branches occur every 8-12 residues |
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Why is glycogen branched?
What is the result? |
Branching allows more monomeric units to be stored in smaller volume since remove OH that would interact with water
↓ groups that can bind H2O, ↑ monomeric units that can be stored |
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How are brancing and catalysis related?
Two things... |
1- Branching creates more ends for catalysis
2- Each branch ends with non-reducing sugar so E can work simultaneously on many branches to remove 1 glucose unit at a time for energy |
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How is glycogen stored?
What % of mass is glycogen in the liver and muscle? |
Large granules with clusters of smaller granules and the enzymes for the synthesis and degradation of glycogen
-10% mass of liver is glycogen -2% mass of muscle is glycogen |
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In the synthesis and metabolism of carbohydrates, why are intermediates frequently phosphorylated?
Three reasons... |
1. Phospho group is relatively stable at pH 7
2. Have negative charge preventing diffusion from cells 3. Serve as important intermediates for subsequent reactions |
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How do enzymes hydrolyze sugars?
Are they specific or not? |
-Enzymes are specific
-Clip at specific linkages |
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What are the two specific enzymes that hydrolyze the storage polysaccharides?
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1. α-amylases for α1-4 linkages (amylose, amylopectin, and glycogen)
2. debranching enzymes for α1-6 linkages (amylopectin and glycogen) |
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Why are starch and glycogen rapidly converted by enzymes?
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Many ends are available for hydrolysis so they have rapid conversion
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What is cellulose?
Where is it found? |
Structural polysaccharide
-Found in: cell walls of plants, stalks, stems, trunks, woody portions of plant tissues, almost the total mass of wood and cotton |
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What is the structure of cellulose?
Two things... |
1. Linear, unbranced homopolymer of 10,000 to 15,000 D-gluocse subunits
2. β 1-4 glycosodic linkage |
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What makes β 1-4 linkage different from α 1-4 linkage?
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α 1-4 configuration results in rigid ring structure (tightly coiled helix)
β 1-4 results in straight, extended chain so all OH groups are available for H-bonding with neighbor |
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Can we digest cellulose?
What can digest cellulose? Aliens? |
NO we can't because we don't have the enzyme for the β 1-4 linkage
-Ruminant animals can digest cellulose b/c have cellulase secreted in stomach by bacteria and protists |
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What is chitin?
Four things... |
1. Structural polysaccharide
2. Hard exoskeleton of insects, arthropods, crustaceans 3. Cell walls of most fungi and algea 4. Second most abundant polysaccharide next to cellulose |
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What is the structure of chitin?
Four more things CHUNKY... |
1. Linear homopolysaccharides of
N-acetyl-D-glucosamine 2. β 1-4 linkages 3. Flat, planar 4. Modification of C2 |
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WTF is a Glycoconjugate?
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Any molecule with carbohydrate on it
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What the hell are Proteoglycans?
Three things... |
1. Complexes of polysaccharides and proteins
2. Macrocmolecules of the cell surface or ECM in which one or more glycosaminoglycan chains are joined covalently to a membrane protein or secreted protein 3. Many possess sulfate: highly negatively charged |
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Where are proteoglycans found?
Four areas... |
1. ECM
2. Cartilage 3. Intervertebral disks 4. Cornea |
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What is the extracellular matrix?
What is it composed of? |
Gel-like substance that fills the space between animal cells
Composed of complex polysaccharides and proteins such as collagen, elastin, fibronectin, and laminin |
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What is the ECM's function and role?
Four things... |
1. Holds cell together
2. Porous pathway for diffusion of nutrients and oxygen 3. Protective function 4. Attached to cell to provide signaling and recognitions |
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What are glycosaminoglycans?
WTF??? Four things... |
1. Repeating disaccharide units
2. Linear 3. Form greater fraction of proteoglycan molecule 4. Often main site of biological activity |
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What are peptidoglycans?
Two things... |
1. Complexes of polysaccharides and peptides
2. Frequently covalently crosslinked to provide rigid protective cover |
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Describe bacteria as an example of a peptidoglycan
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-Bacteria cell wall peptidoglycan alternating β1-4 linkages of N-acetylglucosamine & N-acetylmuramic acid
-Medically significant b/c antigenic components invoke an immune response (response to carbohydrates) -Penicillin inhibits a specific step in peptidoglycan synthesis (vulnerabl to lysis; irreversible inhibitor) |
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What are glycoproteins?
Two things... |
1. Complexes of oligosaccharides with proteins
2. Linkage is either O-linked to serine or threonin residues or N-linked to asparagine residues |
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What are three examples of glycoproteins?
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1. Immunoglobins (based on carbohydrate recognition and signaling)
2. Membrane proteins 3. Hormones |
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What two things provide diversity in glycoproteins?
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1. Oligosaccharide linkages
2. O-linked or N-linked are two examples |
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What does the "sugar code" describe carbohydrates as?
WTF "sugar code" mate? |
-Informational molecules
-Cells use specific oligosaccharides to encode important information about intracellular targeting of proteins, cell-cell interactions, tissue development, and extracellular signals |
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What are lectins?
What do they do? Three chunky things... |
1. Lectins are proteins that bind carbohydrates w/high affinity and high specificity
2. They read the "sugar code" 3. Involved in cell-cell recognition, signaling, and adhesion |
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What type of moieties do some peptide hormones have?
What are some these hormones and there function? |
Oligosaccharide moieties
-Receptor-hormone interaction (mediated by surface oligosaccharides) Lutenizing hormone & thyrotropin: -Regulation of: secretion, uptake, and degradation |
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What importance do oligosaccharides have for animal viruses?
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Animal viruses such as influenza virus attach to host cells via oligosaccharides on the surface
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What are selectins?
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Class of plasma membrane lectins that mediate cell-cell recognition and adhesion in a wide range of cellular processes
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How are selectins involved in the immune system?
Two ways... |
1. Interaction of P-selectin on cells at site of injury with oligosaccharide on surface T-cells
2. Move T-cells through capillary wall at sites of infection or inflammation |
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What are 2 developing applications with carbohydrates?
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1. Carbohydrate vaccines
2. Glycosylation bioengineering in E. coli, yeast, and plants |
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Carbohydrate Vaccines
Four facts... |
1. Agents that stimulate Abs or immune cells to fight disease
2. Historically difficult to isolate from natural sources 3. Historically difficult due to heterogenity and contamination 4. Identify antigenic carbohydrates and synthesis in lab |
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What are 3 examples of synthetic carbohydrate vaccines?
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1. Bacterial menigatic: group c, for
infants 2. Malaria: in development 3. Cancer: globo H for breast cancer (phase I-II), GMK to target melonma, AIDS |
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Glycosylation Bioengineering
and why do I care...? |
50% of human proteins are glycoproteins (but they're heterogenous, glycosylated)
100 enzymes involved in glycosylation |
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What do they do in glycosylation bioengineering?
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Bacterial engineering or budding yeast engineering
-Example: 1/2 of world's insuling supply comes from expression in yeast Goal: get yeast to glycoslate in humans |
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What class of biomolecules do lipids share characteristics with?
Why? |
Carbohydrates
B/c Both have monomeric units |
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Describe Lipids
Four properties... |
1. Highly polymorphic (difficult to define functionally)
2. Water insoluble 3. Either hydrophobic or amphipatic (amphiphillic) 4. Exhibit diversity in structure which in turn results in diversity in function |
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What are four functions of lipids?
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1. Metabolic fuel
2. Structural: membranes & protective coatings 3. Signaling: membrane, hormones, vitamins 4. Transport |
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What are the 6 lipid classifications based on functional groups?
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1. Fatty acids
2. Triacylglycerols & Waxes 3. Glycerophospholipids 4. Sphingolipids 5. Steroids (or sterols) 6. Eicosanoids, ubiquinone, dolichol |
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Which classification(s) are storage lipids?
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-Triacylgylcerols
*storage lipids are neutral |
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Which classification(s) are membrane lipids?
Three of 'em... |
1. Phospholipids
2. Glycolipids 3. Archaebacterial ether lipids *membrane lipids are polar |
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What are the 2 subgroups of phospholipids?
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1. Glycerophospholipids
2. Sphingolipids |
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What are the 2 subgroups of glycolipids?
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1. Sphingolipids
2. Galactolipids (sulfolipids) |
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What is the structure of fatty acids?
Three important things... |
1. Carboxylic acids with hydrocarbon tail
2. (R-COOH) where R is hydrocarbon tail 3. >100 different fatty acids |
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In what four ways do fatty acids differ from one another?
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1. Differ by length of hydrocarbon tail
2. Degree of unsatuaration (# of C=C) 3. Position of C=C 4. A few contain 3-C rings or hydroxyl grops |
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What is the ionization of fatty acids at pH 7?
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pKa for carboxyl group of most FAs is 4.5
Carboxyl group is ionized at pH 7 so negatively charged |
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What is the nomenclature for fatty acids?
What does this mean 12:1(Δ)? |
Numbering begins with carboxyl C as 1
-Chain length is 12 -Number of double bonds is 1 -Position of double bond is between C12 & C1 |
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What are two traits of the most commonly occuring fatty acids?
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1. Even # of C's in unbranched chain due to synthetic pathway b/c of the condensation of acetate
2. Double bonds are cis and seperated by a methylene group |
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What two things does the double bond do to a FA?
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1. Double bonds introduces a rigid bend in the hydrocarbon tail
2. Not free to rotate b/c of the C=C (kink) |
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What two things affect the solubility of FA's?
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1. Chain length & degree of satuaration
2. Longer FA acyl chain and fewer double bonds = lower solubility |
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What does the introduction of double bonds to the FA chain do to the melting point?
Three things... |
1. As the number of double bonds increases, the melting point is lowered
2. Double bonds (unsaturation) disrupts the packing of the FA hydrocarbon tails 3. Less thermal energy to disorder the poorly ordered array |
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What happens to the melting point of saturated FA as length of R↑? Why does this happen?
|
Melting point ↑
The longer the hydrocarbon chain, the more energy that must be invested to disrupt the van der Waals interactions |
|
|
What are the structural implications of straight chain FA tail?
|
1. Free rotation around each C-C
2. Greater flexibility 3. Fully extended is the more stable conformation 4. Minimal steric hinderance 5. Maximize interactions bwt hydrocarbon tails (Van der Waals interactions) 6. Close packing yields crystalline arrays |
|
|
How do saturated and unsaturated fatty acids differ at room temperature?
What does this imply? |
Double bonds have impact on solidity
-Saturated FAs from 12:0 to 24:0 are waxy at room temp -Unsaturated FAs of same chain length are oily |
|
|
What do free FA's only exist in trace amounts in the cell?
Three things... |
1. Most FAs exist as derivatives: ester and amides
2. The loss of O- from carboxyl (lack charged carboxyl group) makes them less water soluble thus need to be bound to be transported 3. In vertebrates, the free FA's that circulate in the bloodstream are bound by serum albumin |
|
|
What are the three most abundant FAs in animals?
|
1. Oleate
2. Palmitate 3. Stearate |
|
|
Which FAs can mammals synthesize and which can they not?
|
Can synthesize saturated and monounsaturated FAs de nove
Require certain polyunsaturated FAs from diet (essential fatty acids) |
|
|
What are triacylglycerols?
Five things... |
1. Simpliest FA derivative
2. 3 FAs, each with ester linkage to glycerol backbone 3. Add FA to each hydroxyl group on glycerol 4. Named after FA's 5. Most naturally occuring have mixed FA's |
|
|
What are the properties of triacylglycerols?
Five more things... ugh! |
1. Nonpolar and hydrophobic
2. Essentially insoluble in water 3. More reduced than sugars 4. Imporant as stored fuel for metabolism 5. Most abundant lipid in mammal by weight |
|
|
Why are triacylglycerols essentially insoluble?
|
Because hydroxyls of glycerol and polar carboxylates of FA's are BOUND IN ESTER LINKAGE
|
|
|
How are lipids with energy storage?
|
-More energy is derived from FA oxidation compared to carbs and proteins
|
|
|
Why is logical to store energy in triacylglycerols?
Three reasons... |
1. Store as nonpolar, hydrophobic compound
2. No added mass due to H20 3. Avoid space and weight by H20 |
|
|
What effect does water solubility have on converting storage molecules to energy?
Two things... |
1. Solubility in water means rapid conversion to energy (ATP)
2. Carbs are converted more rapidly than fats to energy since more water soluble |
|
|
How are triacylglycerols stored in animals?
Three things... |
1. Specialized cells to store large amounts
2. Adipocytes 3. Adiopose tissue provides energy storage, padding, and insulation |
|
|
How are triacylglycerols stored in plants?
|
Seeds
Energy stores with the biosynthetic precursors for germination |
|
|
How do we obtain required FA's from triacylglycerols?
Three things... |
1. Specific hydrolases called lipases
2. Lipases cleave to release FA 3. Digestion occurs in the small intestine |
|
|
What are bile salt? What does it do?
|
Amphipathic derivatives of cholesterol
-Coat fat particles in small intestine → micelles -Enzymes are packed w/in micelle |
|
|
Where are lipases synthesized and activated?
|
Synthesized in pancrease as proenzyme
They are activated in small intestine |
|
|
What specifically do lipases do?
What is the whole cycle that occurs? Five things... |
1. Hydrolyze FA linkages → FA's + monoacylglycerol
2. Transport to intestinal wall in bile-salt micelles 3. FA's & monacylglycerols are absorbed, releasing bile salts 4. Bile salts recirculate in lower part of small intestine, blood, and liver 5. Bound, deliverd to point of metabolism |
|
|
Triacylglycerols and bears...chunky!
Four Reasons... |
1. Energy source
2. Padding 3. Heat 4. Water source (important for hibernating) |
|
|
Describe the typical life of a polar bear?
Four facts... |
1. Eat only a few months of the year
2. Hibernate 3. While hibernating, body temp is close to normal 4. Do not eat, drink, urinate, or defecate during this time |
|
|
What enable polar bears to hibernate through winter?
|
Adaptation
1. Fat oxidation is sole source of energy, heat, and water 2. β-oxidation of FA's → H₂0 + CO₂ |
|
|
Whale story: what is the importance of the spermaceti organ?
|
90% of sperm whale's head is this organ -> very CHUNKY!
Contains 4 tons of spermaceti oil -Aids in sending the directional beam of echolocation clicks which helps in deep dives |
|
|
What is spermaceti oil?
Three things... |
1. Mixture of triacylglycerols & waxes with abundance of unsaturated FAs
2. Liquid at normal whale resting body temp 37C 3. Begins to crystallize at 31C & becomes solid as temp drops few more degrees |
|
|
What is interesting about a sperm's whale dive?
|
Can dive 0.6-2 mi and sit quietly there for 45-60 minutes waiting for squid
*To be able to sit there w/o swimming requires the marine animal to have the same denisty as the surrounding water |
|
|
What enables the sperm whale to sit at depths?
|
-Change buoyancy to match surround of surroundings (denser at deeper depths)
-Adjust density through cyrstallization of spermacetic oil *Congeals at lower temps and thus becomes denser allowing whale to just sit there w/o working |
|
|
How does the sperm whale account for oxygen during deep sea dives?
|
There's a larger blood volume, therefore increased hemoglobin
-Capacity to store O in blood increases -Muscles become enriched in myoglobin |
|
|
How do breaths for sperm whale and land animals compare?
|
-Sperm whale: 80-90% renewal of air
-Land animal: 10-20% renewal of air *Sperm whale has resistance to CO₂ triggering involuntary breathing response |
|
|
What are waxes? What do they do?
Three things... |
1. Waxes are esters of long-chain saturated and unsaturated FA's w/ long-chain alcohols
2. Serve as energy stores and water repellents 3. Melting points are generally higher than triacylglycerols |
|
|
What are five biological waxes?
|
1. Lanolin (lamb's wool)
2. Beeswax 3. Carnauba wax from palm 4. Wax from spermaceti oil 5. Lotions, oilments, and polishes |
|
|
What is the defining characteristic of structural lipids in membranes? What can membrane lipids not be?
|
*Amphipathic* is defining characteristic for membrane lipids
-Nonpolar groups can't be in the lipid bilayer or monolayers -Storage lipids cannot form lipid bilayers |
|
|
What gives membrane lipids their enormous diversity?
|
Enormous diversity b/c of FA tails and polar head groups
|
|
|
Glycerophospholipids
|
-AKA phosphoglycerides
-Most abundant lipid in membranes -Core is Glycerol 3-phosphate |
|
|
What are the properities of glycerophospholipids?
Four properties... |
1. Amphipathic
2. Phosphate group ionized at pH 7 3. X may contribute charge 4. Long, nonpolar hydrocarbon chains |
|
|
Where does the diversity of glycerophospholipids come from?
Three things... |
1. Diversity in structure: architecture & charge
2. FAs 3. X: polar head |
|
|
What does the phosphate group off C₃ contribute to glycerophospholipids?
|
-Joins head group (phosphodiester linkage)
-Bears negative charge at neutral pH |
|
|
What are the enzymes that hydrolyze glycerophospholipids like?
|
Enzymes are very specific
-Specific lipases for a single type bond at a specific location -Example: Phospholipase A₁ for ether linkage of FA and C₁ |
|
|
What problem were food companies trying to overcome with fake fats in real foods?
|
Problem was to replace fat with fake fat and still keep the right taste and consistency
|
|
|
What was Olestra?
|
"WOW Chips"
A true fat -FA's were attached to a core of sucrose *6-8 FAs rather than 3 w/ glycerol -FDA limited use to snacks (junk food) |
|
|
How did Olestra work in the body?
|
Lipases were sterically hindered so couldn't attack extended FA b/c didn't fit specificity (couldn't get into position)
As a result, Olestra passes through small intestine unchanged |
|
|
What were the side effects of Olestra?
|
-Abdominal problems
-When Olestra was micelles going to intestines, everything was going w/ it-Alter uptake of fat soluble vitamins (A,D,E, & K) which normally won't leave body |
|
|
What is the structure of sphingolipids?
Four things... |
1. Polar head and 2 nonpolar tails
2. Sphingosine is core 3. No glycerol core 4. Long chain amino alcohol sphingosine or derivative |
|
|
Ceremide
|
Ceremide is a sphingolipid in which a FA is attached in the amide linkage to the -NH₂ on C-2
X is -H |
|
|
WTF is Sphingomyelin?
Three things... |
1. Contain phosphocholine or phosphoethanolamine as polar head
2. No net charge on polar head 3. Classified along with glycerophospholipids as phospholipids |
|
|
Where are sphingomyelins found?
|
1. Plasma membrane in animal cells
2. Myelin |
|
|
Glycosphingolipids
|
-Head groups have one or more sugars connected directly to the -OH at C-1 of the ceramid moiety
-They do not contain phosphate |
|
|
What are Cerebrosides?
|
Cerebrosides have a singel sugar linked to ceremide
|
|
|
What are uncharged Glycolipids?
Two things...and example?? |
1. Outer face plasma membrane
2. 1-2 sugars attached -Example: Glucosylcerebroside |
|
|
What are Gangliosides?
Four things... |
1. Most complex
2. Have oligosaccharides as polar head groups 3. One or more residues of N-acetylneuraminic acid 4. Negative charge at pH 7 |
|
|
WTF are Globosides?
Two things... |
1. They are neutral glycosphingolipids with two or more sugars
2. Cerebrosides and globosides are sometimes referred to as globosides b/c have no net charge at pH 7 |
|
|
Where are sphingolipids abundant?
Three places... |
1. CNS
2. Major components of myelin sheaths 3. Sphingolipids at cell surface are sites of biological recognition (carbohydrate moiety defines human blood groups) |
|
|
What are the functions of gangliosides?
Three functions... |
1. On cell surface with oligosaccharide extending extracellulary
2. Cell surface markers for cellular recognition & communication 3. Form antigens for blood groups (on surface of RBC) |
|
|
How are lipids affected by enzymes?
Three ways... |
1. Lipids are continually degraded and resynthesized
2. Specific enzyme for each bond 3. Disease states occur b/c of mutations in these enzymes |
|
|
What causes Tay-Sachs disease?
Three things... |
1. Lack of lysosomal enzyme hexosaminidase A (one enzyme is defected)
2. Specific ganglioside accumulates in the brain & spleen 3. Accumulation of partly degraded gangliosides is toxic |
|
|
What are the three symptoms of Tay-Sachs disease?
|
1. Degeneration of nervous system
2. Progressive retardation in development 3. Paralysis, blindness, and death by age 3 or 4 |
|
|
What is Tay-Sachs disease?
Dominant or recessive? |
Human genetic disease
*Defective gene is RECESSIVE Can measure levels hexosaminidase in skin cells of parents to detect carriers) |
|
|
What are four common diseases of lipid storages or sphingolipids?
|
1. Tay-Sachs
2. Gaucher's 3. Fabry's 4. Niemann-Pick |
|
|
What are five common features of lipid diseases?
|
1. Usually only a single sphingolipid accumulates
2. Stored lipid is structurally OK 3. Rate of biosynthesis of accumulating lipid is normal 4. Specific catabolic enzyme is deficient in each disease 5. Extent of enzyme deficiency is the same in all tissues |
|
|
What are sterols?
|
Sterols are structural lipids in membranes
-Cholestrol characteristic animal membranes -Some plant & fungi specific sterols *Not found in prokaryotes |
|
|
What is the characteristic structure of sterols?
Where does their diversity come from? |
4 fused rings
-Less flexible relative to other lipids -Diversity is generated by length of side chain & polar head groups |
|
|
What three things do sterols do to the fluidity of membranes?
|
1. Modulate the fluidity of membranes
2. Decrease fluidity b/c less flexible 3. Cannot form bilayer alone |
|
|
Why are sterol compounds biologically important?
Three reasons... |
1. Important for specific activities
2. Steroid hormones regulate gene expression 3. Bile acids (derivatives of cholesterol) act as detergents |
|
|
What are 4 lipids that act as signaling molecules?
|
1. Phosphatidylinositols & derivatives involved in intracellular signaling
2. Sphingosine derivatives 3. Eicosanoids 4. Vitamins |
|
|
How do sphingosine derivatives act at signaling molecules?
Two ways... |
1. Ceremide and sphingomyelin are potent regulators of protein kinases
2. Involved in regulation of cell cycle, differentiation, cell migration, and programmed cell death (apoptosis) |
|
|
How do eicosanoids act as signaling molecules?
Five ways... |
1. Are not transported
2. Act at site were synthesized 3. Involved in reproductive function 4. Inflammation, fever, pain associated with injury 5. Prostaglandins, thromboxanes, leukotrienes |
|
|
How do vitamins act as signaling molecules?
Two things... |
1. Vitamins A & D are hormone precursors
2. Vitamins E & K are oxidation-reduction cofactors |
|
|
What are passive role lipids?
|
MEMBRANE LIPIDS
Lipid that are used as fuels and are stored until oxidation |
|
|
What are active role lipids?
Four facts... |
1. Metabolites/messengers
2. Potent signals (hormones) 3. Enzyme cofactors in e- transfer chain 4. Pigment molecules |
|
|
What are the polar and nonpolar regions of triacylglycerols?
|
-The whole molecule is nonpolar
-FA carboxylic acid groups are esterfied to glycerol's -OH group |
|
|
What are the polar and nonpolar regions of glycerophospholipids?
|
-Phosphate groups and attached head groups are polar
-FA tails are nonpolar |
|
|
What are the polar and nonpolar regions of sphingolipids?
|
POLAR:
-OH group on alcohol sphinogsine is polar -Some have phosphate and/or sugars both of which are polar NONPOLAR: FA's tails are nonpolar |
|
|
What are the polar and nonpolar regions of ganglioside?
|
POLAR:
Carbohydrate moiety is polar NONPOLAR: FA's tails are nonpolar |
|
|
What are the polar and nonpolar regions of cholesterol?
|
POLAR:
The -OH group is polar NONPOLAR: Steroid nucleus and acyl chain are nonpolar |
|
|
Why does adding sodium hydroxide to grease traps help clean them?
Three facts... |
1. Triacylglycerols are hydrolyzed by the NaOH to for glycerol and the sodium salts of the free fatty acids
2. FA's from micelles 3. Process is called saponification |
|
|
Describe how chromatographic separation of lipids work?
What elutes first? Three facts... |
Silica gel (POLAR) is used
1. Most hydrophobic lipids elute first since they don't interact with gel 2. Most hydrophilic elute last since they bind tightly w/ gel 3. Polar lipids elute in order of increasing polarity by washing column w/ solvents of progressively higher polarity |
|
|
What is the lipid bilayer?
What do they contain? How thick are they? |
Biological membrane
All contain proteins (trilaminar appearance) 5-6 nm w/o proteins (thinner) 5-8 nm (50-80 A) w/proteins (not as dense) |
|
|
What five complex functions are associated with biological membranes?
|
1. Barrier
2. Selective transport 3. Receptors for signaling 4. Respiratory e- transport chain enzymes 5. Myelin of nerve fibers (modified to insulate) |
|
|
Why is "flip-flop" (transbilayer) diffusion not favorable for lipid membranes?
Three reasons... |
1. Very slow
2. Polar head has to flip through hydrophobic bilayer 3. Energetically unfavorable (high energy needed for flip to occur) |
|
|
When can transverse ("flip-flop") diffusion occur?
Three situations... |
1. Can occur if catalyzed by flippase
2. Flippase is coupled to ATP turnover 3. Flippase sequesters polar head group which doesn't like nonpolar region |
|
|
What is the preferred way for lipids and proteins to move in the bilayer?
Three things... |
1. Lateral diffusion
2. Change places with neighbor lipid molecule 3. Rapid and energetically favorable |
|
|
What did the Frye-Edidin experiment make?
Two things... |
1. Made antibodies to surface antigens
2. Antibodies were specific for either mouse or human antigens |
|
|
How could they tell if the antibody was mouse or human?
Two ways... |
1. Detect mouse by fluorescein-Ab (yellow/green color)
2. Detect human by rhodamine-Ab (red color) |
|
|
What did Frye & Edidin do once the antibodies were formed?
Three things... |
1. Formed heterokaryon (combination of 2 cells)
2. Red & green markers were intermixed 3. They asked what would happen to murine & human surface markers |
|
|
What did Frye & Edidin see happen to the markers?
|
Even distribution at first
Then.. As a function of time at 37⁰C the markers began to mix |
|
|
What were the first two hypotheses used to explain Frye-Edidin's results?
|
1. Very rapid turnover of membrane antigens
2. Ag from cytoplasmic pool incorporated into membrane (localized, still have 50/50 split) |
|
|
What were the second two hypothesis used to explain Frye-Edidin's results?
|
3. Movement of membrane Ag cytoplasmic pool & back again (internalize then go back up to surface at different location)
4. Diffusion of Ag in the plane of the membrane |
|
|
Frye-Edidin:
What experiment was done to address hypothesis 1? What happened? |
EXPT: Inhibited protein synthesis. Used puromycin, cycloheximide, & chloramphenicol as inhibitors
*Results they saw were affected so was not this hypothesis* |
|
|
Frye-Edidin:
What experiment was done to address hypothesis 2? What happened? |
EXPT:Inhibited ATP formation using NaF (dinitrophenol)
*Depleted cell of energy and still saw same fluorescent pattern* |
|
|
Frye-Edidin:
What experiment was done to address hypothesis 3? |
EXPT: Metabolic suppression by lowering temperature
|
|
|
What did Frye & Edidin want to find in their experiments?
|
EVIDENCE that Ag moved laterally
|
|
|
What three conclusions did Frye & Edidin come to?
|
1. Cell surface is NOT a rigid structure
2. Membrane's fluid enough to allow free diffusion of surface markers 3. Direct evidence that there is a temporal factor to movement |
|
|
What role does temperature play in movement?
Five roles... |
1. Temperature dependent
2. Fluidity is controlled by temperature 3. Colder temp = less fluid 4. At low temps, clear 50/50 split of markers 5. At higher temps, more & more mixing of markers |
|
|
Who came up with the fluid mosaic model?
Two douches... |
S. Jonathan Singer
& Garth L. Nicolson |
|
|
What 3 types of lipid aggregates can form when amphipathic lipids are mixed with water?
|
1. Micelles
2. Open bilayer 3. Closed bilayer |
|
|
How are lipids in micelles arranged?
Three facts... |
1. Hydrophobic chains of FA are sequestered at core of sphere
2. Virtually no water in hydrophobic core 3. Hydrophilic head groups are at the surface |
|
|
How are lipids in open bilayers arranged?
|
All acyl side chains except those at the edges of the sheet are protected from interaction w/ H₂0
|
|
|
What is a bilyaer?
Two things... |
1. Lipid aggregation in H₂0 in which 2 lipid monolayers form a 2-dimensional sheet
2. Bilary formation occurs most readily when the cross-sectional areas of the head group and acyl side chain(s) are similar |
|
|
What is the downfall of open bilayers?
Two problems... |
1. Hydrophobic regions at its edges are transiently in contact w/ H₂0
2. The bilayer sheet is relatively unstable and spontaneously forms a third aggregate (liposome) |
|
|
How does a closed bilayer form?
|
A 2-D bilayer folds on itself to form a closed bilayer
|
|
|
What is a closed bilayer?
What is the advantage of closed over open bilayer? |
A closed bilayer is a 3-D hollow vesicle (liposome) that encloses an aqueous cavity
*By forming vesicles, bilayers lose their hydrophobic edge regions and achieve max stability in an aq. environment |
|
|
What does the fluid mosaic model state?
Three statements... |
1. Membrane is dynamic
2. lateral diffusion of both proteins & lipids 3. Membrane mosaic is fluid b/c most of the interactions are non-covalent |
|
|
What 2 types of noncovalent interactions is the fluid mosaic model based upon?
|
1. Hydrophobic & hydrophilic
and 2. Sequestering hydrophobic domains away from H₂0 |
|
|
What is the structure of the membrane bilayer?
Four things... |
1. Phospholipids form a bilayer
2. Nonpolar regions of lipid molecules face the core of the bilayer 3. Polar head groups face outward interacting w/ aq phase on either side 4. Proteins are embedded in this bilayer sheet & are held by hydrophobic interactions between membrane lipids & hydrophobic domains in the proteins |
|
|
Where did evidence for the fluid mosaic model come from?
Two places... |
1. Fry-Edidin expts
2. Freeze-fracture w/ electron microscopy |
|
|
What evidence came from freeze-fracture with electron microscopy to support the fluid mosaic model?
|
Structural evidence
*Argued for membrane being bilayer & fluid |
|
|
What was done in freeze-fracture with electron microscopy?
What was seen? |
Froze cells that proteins associate w/ and split cell (split bilayer)
*Saw protein parts poking out from split bilayers |
|
|
What are the 3 classes of membrane proteins?
What are they based upon? |
1. Integral or Intrinsic
2. Peripheral 3. Lipid-anchored membrane proteins * based upon on how you separate them |
|
|
What are Integral or Intrinsic Membrane Proteins
Three things... |
1. Transmembrane or in hydrophobic domain
2. None are completely buried in lipid bilayer 3. Firmly associated w/ the membrane |
|
|
How do you remove integral membrane proteins?
Example? |
Remove w/ agents that interfere w/ hydrophobic interactions
*Use mild detergents (make membrane soluble), organic solvents, or denaturants |
|
|
What are Peripheral Membrane Proteins?
Three facts... |
1. Not covalently bound
2. Interact w/ one face via electrostatic interactions 3. Also H-bond w/ hydrophilic domains of integral proteins & w/ polar head groups of membrane lipids |
|
|
How do you remove peripheral membrane proteins?
What would you use? |
Disrupt ionic interactions
Use salt, change pH, carbonate at high pH, add urea, or removal of Ca2+ |
|
|
What are Lipid-Anchored Membrane Proteins?
|
Protein tethered to membrane by covalent linkage to lipid
*Found in viruses & eukaryotic cells |
|
|
From using what type of cell does our initial understanding of membrane proteins come from?
|
Studies using human erythrocyte or RBC
|
|
|
Why use RBC for studying membrane proteins?
Three reasons... |
1. Easy to obtain in large amounts
2. No membrane-bound organelles 3. Experimentally easy to manipulate |
|
|
What have X-ray crystallography and NMR contributed to our understanding of membranes?
Two things... |
1. Provided structural information
2. Technically challenging but a significant # of membrane channels & proteins have been crystallized and their structures determined |
|
|
What holds integral membrane proteins in the membrane?
Three reasons... |
1. Hydrophobic interactions w/ lipids
2. Some have singe hydrophobic sequence (glycophorin) 3. Some have multiple hydrophobic sequences (bacteriorhodopsin) |
|
|
Describe the hydrophobic sequences that hold integral proteins in the membrane?
Four facts... |
1. Each sequence is α-helical (usually of 6 to 7 turns)
2. Enough sequences to span entire membrane 3. One hydrophilic domain is on the outer surface 4. One hydrophobic domain protrudes from the inner face of the membrane |
|
|
What is a common motif in membrane proteins involved in signal reception?
|
*7 hydrophobic membrane-spanning helices*
|
|
|
Describe Bacteriorhodospin that is covalently bound to retinal
Five facts... |
1. 247 aa protein
2. Integral membrane protein 3. Salt loving 4. Light driven proton pump - ATP 5. Light absorbing group |
|
|
Describe the structure of bacteriorhodospin
Four parts... |
1. 7 α-helical segment
2. Approximately 25 aa in each α-helical segment 3. Polar residues near surface 4. Hydrophobic residues w/ membrane bilayer |
|
|
How can the topology of an integral membrane protein be predicted?
Is it easy to obtain? |
Predict from aa sequence
-Sequence info is easy to obtain but relatively few 3-D structures have been obtained by NMR or X-ray crystallography |
|
|
What predictions have been made about integral membrane proteins based on aa sequence?
Three predictions... |
1. 20 aa stretch of hydrophobic residues indicative of transmembrane domain
2. An α-helical sequence of 20-25 aa is just long enough to span the lipid bilayer (0.15 nm/aa) 3. Hydrophobic interactions between aa R-groups & lipids stabilize protein membrane |
|
|
What is the Hydropathy Index?
|
Used to predict whether an AA stretch is transmembrane
|
|
|
How does a hydropahty index work?
|
Relative polarity of each aa is determined experimentally by measuring free energy change w/ movement from hydrophobic solvent into water
|
|
|
What do the free energy changes in a hydropathy index indicate?
Three things... |
1. Charged, polar aa: exergonic (release free energy)
2. Aromatic or aliphatic hydrocarbon aa: endergonic 3. Hydrophoic indicates a region that would cross membrane |
|
|
What two amino acid(s) are characteristic of transmembrane proteins?
Why? |
Tyr & Trp residues
-They're at the interface between lipid and water -Side chains appear to anchor protein in membrane b/c can act simultaneously w/ central lipid phase & aq phase on either side of membrane -Also prevent H₂0 from seeping around protein & creating channel on sides |
|
|
Where are Tyr and Trp found?
Where are charged residues found? |
Try & Trp found where nonpolar acyl chains meet polar head group
Charged residues (Lys, Arg, Glu, Asp) are found exclusively in aq phase |
|
|
What will a polypeptide chain do when it's surrounded by lipids, and has no water molecules to interact with do?
Three things... |
1. Form α helices or β sheets
2. Intrachain H-bonding is maximized 3. If the side chains of all aa in a helix are nonpolar, hydrophobic interactions w/ the surrounding lipids further stabilize the helix |
|
|
What other conformations besides
α-helices can transmembrane proteins exhibit? Four conformations... |
1. β barrel
2. β sheet 3. Just seven to nine residues of β-conformations are needed to span a membrane 4. Can form channel using β-conformations |
|
|
What are β-barrels motifs in membrane proteins?
Two things... |
1. 20 or more transmembrane segments form β sheets that line a cylinder
2. Barrels allow all possible H-bonds where as planar β sheets do not max these interactions and are generally not found in the membrane interior |
|
|
What interactions stabilize β-conformations?
Three interactions... |
1. H-bonding
2. Every second residue in membrane-spanning segment is hydrophobic and interacts w/ the lipid bilayer 3. Aromatic side chains are commonly found at the lipid-protein interface |
|
|
What anchors membrane proteins to the bilayer?
Three things... |
1. Covalently attached lipids anchor membrane proteins
2. Attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer & holds the protein at the membrane surface 3. Many proteins have more than one attached lipid moiety |
|
|
What other interactions contribute to the stability of the attachment?
|
Ionic attractions between positively charged Lys residues in the protein
and negatively charged lipid head groups |
|
|
Besides anchoring a protein to the membrane, what other roles do attached lipids have?
Three other roles... |
1. Frequently a specific role in signaling function
2. GPI anchors on outer face and only w/in clusters 3. Fernesyl groups on inner face exclusively |
|
|
What three functions do integral proteins (Integrins) have?
|
1. Mediate cell-cell or cell-ECM interactions and adhesions
2. Cell-cell communication 3. Internal/external communication |
|
|
What are 4 examples of integral protein types that function in cell-cell interactions
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1. Integrin: ligand-binding region
2. Cadherin: Adhesive domian 3. N-CAM: immunoglobin-like domians 4. Selectin: lectin domian (binds carbs) |
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What are Integrins?
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They are heterodimeric proteins anchored to the plasma membrane by a single hydrophobic transmembrane helix in each subunit
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Describe two structural things about integrins...
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1. Consist of α & β transmembrane polypeptides
2. Their large extracellular domains combine to form specific binding sites for extracellular proteins such as collagen & fibronectin |
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What three things are integrins involved in?
|
1. Adhesion
2. Signal transduction 3. Regulate platelet aggregation at site of wound, tissue repair, activity of immune cells, invasion by tumor |
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What the hell do selectins do?
Three things Chunky.... |
1. Have extracellular domains for Ca+ binding
2. Present in primarily in various types of blood cells (essential in blood-clotting process) 3. CHO binding essential for blood clotting |
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Describe the movement of various particles across the synthetic phospholipid bilayer...fun!
Six types of particles... |
1. Gases (CO₂, N₂, O₂): diffuse across easily
2. Small, uncharged polar molecules (urea, ethanol): diffuse across easily 3. Water: critical channels 4. Large, uncharged polar molecules (glucose): can't diffuse 5. Ions: can't diffuse 6. Charged, polar molecules: can't diffuse |
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What is simple diffusion?
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-Solute moves from region of high to low concentration until two compartments have equal solute concentrations
-Equalize |
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What is the transmembrane electrical gradient?
Why does it happen? |
Membrane potential Vm
-When ions of opposite charge are separated by a membrane, there's a transmembrane electrical gradient |
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What does this membrane potential Vm produce?
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A force that:
-Opposes ion movements in order to increase Vm or -Drives ion movements to reduce Vm |
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What two things does spontaneous movement of charged solute depend upon?
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1. Concentration (chemical gradient)
and 2. Electrical gradient (Vm) |
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What is the electrochemical gradient?
What does it consist? Two things... |
AKA electrochemical potential
-Consists of chemical gradients and electrical gradients |
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How is the behavior of solutes during diffusion in accordance with the second law of thermodynamics?
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Molecules tend to spontaneously assume the distribution of greatest randomness and lowest energy
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How is randomness produced in diffusion of polar or uncharged solutes through a lipid bilayer?
Three things... |
1. Solute must give up interactions w/ water molecules in hydration shell
2. Increase in entropy 3. Energy used to strip away hydration shell and move polar compound from water into & through lipid is regained as compound leaves membrane on other side and is rehydrated |
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Describe the free energy change associated with hydrophilic solutes passing across a membrane
Two things... |
1. Simple diffusion ▵G is so high for translocation of a polar solute that lipid bilayer is impermeable
2. Membrane proteins lower energy of activation ▵G by providing alternative path through bilayer for specific solutes |
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What is Facilitated Diffusion?
|
AKA Passive Diffusion
-Diffusion of polar substance across biological membrane through a protein transporter |
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What are Transporters?
Two things... |
AKA Permeases
1. Membrane proteins that speed the movement of a solute across a membrane by facilitating diffusion 2. They differ from the usual enzyme in that "substrates" are moved from one compartment to another but are NOT chemically altered |
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What are the two classifications of transporters?
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1. Carriers
2. Channels or pores |
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What do carriers do?
What are three characteristics of carriers? |
Carriers bind substrate with high stereospecificity
1. Catalyze transport 2. Are saturable (in the same sense as enzymes are) 3. Many are monomer proteins |
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What are the two subgroups of carriers?
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1. Secondary active transporters
and 2. Uniporters |
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How do the rates of channels/pores compare with those of carriers?
Two things... |
1. Generally allow movement at rates several orders of magnitude greater than those of carriers
2. Rates approach the limit of unhindered diffusion |
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What are three characteristics of channels or pores?
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1. Can be weakly selective or nonspecific
2. Not saturable, pore may always be open 3. Most are oligomeric proteins |
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What does the glucose transporter do?
Three facts... |
1. Mediates passive transport of glucose from plasma
2. Glucose enters RBC via facilitated diffusion 3. Rate of transfer 50,000 times greater than by simple diffusion |
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What is the specific glucose transporter?
Four things... |
1. GLUT 1
2. Integral membrane protein 3. Specific for D-glucose 4. Structure not yet determined |
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What are two characteristics of transport by GLUT 1?
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1. Saturable
2. Not ATP dependent |
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Why is transport by GLUT 1 saturable?
Two reasons... |
1. Binding to specific site that causes a conformational change
2. Limited # of proteins in membrane so saturation depends on [Glucose] |
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What are the four steps of glucose transport?
|
1. Glucose in blood plasma binds to a stereospecific site on T₁; lower activation energy
2. Conformational change from S(out)·T₁ to S(in)·T₂ 3. Glucose is released from T₂ into cytoplasm 4. Transporter returns to T₁ conformation |
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What are GLUT 1 Transport Kinetics?
Two things... |
1. Kt is concentration of glucose at which have 50% max rate
2. 12 glucose transporters have kinetic differences in their rate of transfer and Kt |
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What is the Chloride-Bicarbonate exchanger in RBC?
What is its role? |
AKA Anion Exchange (AE) Protein
-Cotransport system that allows entry and exit of HCO₃⁻ w/o changing transmembrane Vm *Its role is to increase the CO₂ carrying capacity of blood |
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What is the AE protein?
Two facts... |
1. Integral membrane protein (Anion exchange protein)
2. Spans membrane at least 12 times |
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What does the AE protein facilitate?
Four things.. |
1. Facilitated diffusion
2. Antiport cotransport 3. For each HCO₃⁻ ion that moves in one directions, one Cl⁻ ion moves in the opposite direction 4. Thus, no net transfer of charge |
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What happens in the absence of chloride to the bicarbonate transport system?
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It Stops.
The coupling of HCO₃⁻ and Cl⁻ movements is necessary |
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What are Cotransport Systems?
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Systems that simultaneously carry two solutes across a membrane
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Differences between Antiport vs Symport?
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Antiport: two substrates move in opposite direction
Symport: two substrates move in same direction |
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What are Uniport Systems?
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Transporters that carry only one substrate such as GLUT 1
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What is active transport?
What does it require? |
Movement of solute AGAINST chemical or electrochemical gradient which results in the accumulation of a solute above the equilibrium point
-Its endergonic *Requires energy to move solute against a concentration or electrochemical gradient |
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What are the two types of active transport?
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1. Primary active transport
and 2. Secondary active transport |
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What is Primary Active Transport?
Examples? |
Solute movement is coupled directly to an exergonic chemical reaction (usually ATP turnover)
-Example: Na⁺K⁺ ATPase |
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What is Secondary Active Transport?
Examples? |
Energy required for the solute movement is obtained from another primary transport system
-Examples: Na⁺-glucose symport; Na⁺ gradient established by the Na⁺K⁺ ATPase |
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What are the five steps in the mechanism of the Na⁺K⁺ ATPases?
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1. Transporter binds 3 Na⁺ from inside of the cell
2. Phosphorylation favors P-EnzII 3. Transporter release 3 Na⁺ to the outside and binds 2 K⁺ from outside of cell 4. Dephosphorylation favors EnzI 5. Transporter release 2 K⁺ to inside |
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What two forms does the ATPase cycle between?
|
1. Phosphorlylated form (P-EnzII) with high affinity for K⁺ and low affinity for Na⁺
2. Dephosphorylated form (EnzI) with high affinity for Na⁺ and low affinity for K⁺ |
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WTF are P-type ATPases???
|
P-type ATPases are Active transporters
ATP-driven cation transport that is reversibly phosphorylated by ATP as part of the transport cycle |
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What is the result of Na⁺K⁺ ATPase?
Three results... |
1 ATP
2 K⁺ in 3 Na⁺ out |
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Why is active transport necessary for Na⁺ and K⁺ movement?
Three reasons... |
1. Na concentration is lower in cell than surrounding
2. K concentration is higher is cell than surroundings 3. Movement against electrochemical gradient is coupled to ATP conversion to ADP and Pi (phosphate transfer to protein) |
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What three things does the specific inhibitor Oubain do to the Na+K+ ATPase?
|
1. Bind to the form of the enzyme that is open to the extracellular side
2. Locks 2 Na+ ions 3. Prevents change conformation necessary to ion transport |
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What is Oubain?
Structure? |
Inhibitor to Na+K+ ATPase
Steroid (4 rings fused structure) |
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What secondary active transport is linked with Na+K+ ATPase?
Why? |
Glucose and certain AA's are co-transported with Na+
-If one didn’t have the Na+ gradient, these things would not be transported |
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How are membrane transporters pharmaceutical targets?
Two ways... |
1. Target bacterial membrane transporters with inhibitors
2. Kill microbial cells by disrupting transport processes |
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What properties of lipids are responsible for bilayer formation?
Four properties... |
1. Amphipathic
2. Form 2-D sheets with hydrophilic domains exposed to water and hydrophobic regions buried in interior 3. Close upon themselves to avoid exposing hydrophobic edges to water 4. Hydrophobic interactions are driving force for membrane formation |
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How do membranes at lower temperatures maintain fluidity?
Two things... |
1. Membranes contain a higher percentage of polyunsaturated fatty acids
2. Higher content of unsaturated FA's lowers the melting pt. of lipid mixtures |
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At pH 7, TRY crosses a lipid bilayer one-thousandth the rate of closely related substance indole? Why?
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TRY exists as zwitterions at pH 7 having both positive and negative charge
*Indole is uncharged, thus movement of less polar indole through hydrophobic membrane is more energetically favored |
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What is nanomedicine?
Two facts... |
1. An offshoot of nanotechnology
2. Highly specific medical intervention at the molecular scale for curing diseases or repairing tissue |
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What did Peter Agre discover that won him the Nobel Prize in (03)?
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Water channels
*H20 entry into and out of cells |
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What did Roderick MacKinnon discover that won him the Nobel Prize in (03)?
|
Structural and mechanistic studies of ion channels (K+ channel)
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What are aquaporins?
What do they do? |
Water channels (transmembrane)
They allow rapid movement of water molecules and are freely permeated by H20 but NOT H30+ |
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How were aquaporins discovered?
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Discovered when purified the protein AQP-1
Discovered accidentally when working on RBC membrane |
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How do aquaporin channels work?
Four things... |
1. High capacity for H2O not H3O
2. Directed by osmotic gradient 3. Movement is continuous stream 4. Reversibly inhibited by Hg++ |
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What is the structure of AQP 1?
Four things.... |
1. Tetramer of 4 identical monomers
2. Each subunit has channel (4 channels total) 3. NPA sequence forms the channels of the pore permitting water and excluding charged ions |
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What is the NPA sequence?
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Asn-Pro-Ala
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Describe the structure of a single AQP-1 subunit..
Three important things... |
1. AA residues that line channel are generally nonpolar BUT Carbonyl O project into channel at intervals to H-bond with water and permits passage
2. At the constriction point there is a Phe58 that acts as gate keeper b/c it's hydrophobic 3. Asn76 & Asn192 in NPA loops H-bond also |
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What two roles do Arg and His have in aquaporins?
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1. Critical residues
2. Repel any protons that might leak in |
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What are five clinical disorders that aquaporins are implicated in?
|
1. Renal-vascular disease
2. Brain injury & edema 3. Loss of vision 4. Starvation 5. Thermal stress |
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What is CFTR?
|
Cystic Fibrosis Transmembrane Conductance Regulator
*Cl- channel -Nonfunctional chloride ion channel is genetic disease (severity depends on where aa is missing) |
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What four approaches does Erik Jackobson use to engineer channels and pores?
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1. Redesign existing structure
2. De nove design 3. In vitro evolution/combinational approaches 4. Introduction of unnatural AA’s |
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What scaffold is often chosen?
Four reasons why? |
*β Barrel favors redesign*
1. It has an open interior meaning few significant side-chain interactions 2. Pore stays open, no spontaneous gating 3. Robust, easy to express 4. Engineering of α helices is harder b/c open to environment |
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What are two ways to go about redesigning an existing structure?
|
1.Start with something complex and change it
2. Start with something simple and build function into it |
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How did Chris Schafmeister use chemistry to engineer pores?
Three facts... |
1. Synthetic chemistry to generate building blocks
2. Thoughtful assembly and protein folding 3. Used chemistry to design what he wanted |
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How did Hong Koo-Kim impact instrument development for nanomedicine? (two ways)
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1. Developed nanofabrication and nan-optics technology
2. Measure the forces and motion produced by mitotic motors |
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What are four challenges of nano-medicine
|
1. Requires multidisciplinary teams of scientists and engineers
2. Integration of experimental & computational approaches 3. Development of biomimetic systems at nanoscale level 4. Fabrication of nanoscale materials and devices that can be integrated into biological environments |
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What are the four major types of biochemical reactions?
|
1. Oxidation-Reduction
2. Carbon-carbon bond formation/breakage involving carbonyl groups 3. Internal re-arrangements/eliminations involving C=C bonds 4. Group transfers |
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What are oxidation-reduction reactions?
Three parts... |
1. Transfer of electrons
2. One compound is oxidized by either removing protons (and their e-) or adding oxygen 3. One compound is reduced by either adding protons (and their e-) or removing oxygen |
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What is Oxidation?
|
Lose e-
or Add O |
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What is Reduction?
|
Gain e-
or Remove O |
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What are the five oxidation states of carbon?
|
1. Alkane (Most REDUCED)
2. Alcohol 3. Aldehyde (Ketone) 4. Carboxylic Acid 5. Carbon Dioxide (Most OXIDIZED) |
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What two enzymes catalyze
oxidation-reduction reactions? |
1. Dehydrogenases
2. Oxgenases |
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What is a Dehydrogenase?
|
AKA Oxidoreductases
*They remove 2 e- and 2 protons and commonly transfer these to the electron acceptors NAD+ or FAD |
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What are Oxygenases?
|
AKA Oxidases
Use oxygen as an e- acceptor |
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How do oxygenases and oxidases differ?
|
Oxygenases incorporate one or both oxygen molecules into the product
*Oxidases DON'T incorporate the oxygen into the product (usually make H2O) |
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In a carbonyl carbon, how is the charged distributed?
Three facts... |
1. Partial negative charge on O (potential nucleophilic site)
2. Partial positive charge on C (potential electrophilic site) 3. Strong electron withdrawaling properties of O polarize (weaken) the C=O bond |
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Arrange the element H, S, C, N, O in order of increasing electronegativity
|
H > C > S > N > O
Least electronegative: H Most electronegative : O |
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How is the alpha carbon affected by carbonyl groups?
Three ways... |
1. Carbonyl groups facilitate nucleophile formation on the alpha carbon
2. Proton on alpha carbon is acidic so it is easily removed to form an enolate 3. Enolate loses H+ to form a carbanion |
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Electrophile vs Nucleophile
|
Electrophile is e- deficient
Nucleophile is e- rich |
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What is an example of C-C formation?
What is an example of C-C bond cleavage? |
Aldol condensation is C-C bond formation
Decarboxylation is C-C bond cleavage |
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How do isomerizations and eliminations proceed?
Four things... |
1. Proceed through C=C
2. Elimination removes H2O to form C=C 3. Isomerization adds H2O and breaks C=C 4. End result is moved functional group from one C to another |
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What is a group transfer?
|
A simple nucleophilic displacement
*Have leaving group displaced by nucleophile |
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What are five common nucleophiles?
|
1. Water (hydrolysis: break ester or amide bond with H2O)
2. Inorganic Phosphate (phosphorylation) 3. Phosphate (from ATP; phosphorylation converts OH to PO4) 4. Alcohols (ester formation COOH -> COOR) 5. Amines (amide formation COOH -> COONR) |
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What determines if a biochemical reaction occurs?
|
ΔG
-ΔG: reaction will proceed +ΔG: reaction will not proceed *ΔG only tells if reaction can happen, not how fast it will proceed |
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What is the formula for ΔG?
|
ΔG = ΔH -TΔS
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What is the difference between ΔG and ΔG’°?
|
ΔG: is the actual free energy change that occurs under experimental conditions
ΔG’° is the free energy change listed in tables that occurs under standard conditions |
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What are standard conditions?
Three things... |
1. pH =7
2. H2O = 55M 3. [Mg++] = 1mM |
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How is ΔG’° derived?
|
Derived from the equilibrium constant:
ΔG’°= -RTlnK'eq ΔG’° represents the free energy to reach equilibrium when starting from standard conditions |
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How does ATP provide energy?
Three ways... |
1. Provides energy by group transfer NOT hydrolysis
2. Often makes poor leaving group into good one 3. Couples unfavorable reactions (not in parallel with them) |
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How is the mass action ratio (Q) calculated?
|
Same way as equilibrium constant except it reflects the actual starting concentrations of reactants and products
*Q is under actual conditions |
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How do you calculate the real ΔG from ΔG’°?
|
ΔG = ΔG’° + RTlnQ
*if have standard conditions ΔG=ΔG’° (drop RTlnQ portion of equation b/c Q would = 0) |
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If ΔG is positive does that mean that the reaction can never happen?
|
This Only means it won’t happen under the initial reaction conditions you have chosen
If you change the reaction conditions appropriately you can get the reaction to occur |
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How can you change a +ΔG to a -ΔG?
Two ways... |
1. Change lnQ to make it sufficiently negative
2. Change Q by changing ratio of products: reactants |
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In what two ways can you change Q?
|
1. Increase the concentration of Starting Material
or 2. Decrease the concentration of products |
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Besides changing Q, how else can you change a +ΔG to a -ΔG?
|
Couple a favorable reaction to an unfavorable reaction
*Since free energy changes are additive, the total reaction will have a -ΔG |
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What is ATP?
What three things is it composed of? |
Adenosine Tri-phosphate
-Holds genetic information and high energy molecule *Composed of: 1. base (adenine) 2. sugar 3. 3 phosphates (2 anhydrides) |
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Why is ATP used to drive chemical reactions?
Two reasons... |
1. It is a High energy compound
2. The products of ATP hydrolysis are more stable than ATP |
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What type of reaction is ATP hydrolysis?
|
*Group transfer*
-Transfer phosphate group -Use H2O to split |
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What is considered “high energy”?
|
Anything with a ΔG’° more negative than 25 kJ/Mol
< -25 kJ/Mol |
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What are four “high energy” compounds?
|
1. Acetyl-CoA
2. ATP (->AMP + PPi) 3. ATP (-> ADP + Pi) 4. ADP (-> AMP + Pi) |
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If the ΔG for ATP hydrolysis is so large and negative, why is ATP chemically stable?
|
Remember ΔG only evaluates if reaction will proceed, not how fast**
The speed of the reaction is dictated by activation energy and the AE for ATP hydrolysis at pH 7 is LARGE |
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Where does ATP come from?
Two places... |
1. Metabolism (oxidative phosphorylation)
2. Gentle release of energy |
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What is the electromotive force?
|
The amount of work produced in oxidation/reduction reaction
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What is the reduction potential ΔE?
|
The relative affinity of an electron acceptor for electrons
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|
What is the standard reduction potential ΔE’°?
|
For each half reaction in a redox reaction, the standard redox potential measures the affinity of the e- acceptor for e- under standard conditions
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What two things does the value of ΔE’° indicate about the species?
|
1. The more positive the ΔE’°, the more affinity the species has for an e-
2. If a species has -ΔE’° then the back reaction would go (be oxidized not reduced) |
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What must the values of ΔE and ΔG be for a reaction to be spontaneous?
|
-ΔG
+ΔE |
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How do ΔE and ΔE’°differ?
|
ΔE’° describes redox potential under STANDARD conditions
ΔE describes redox potential under EXPERIMENTAL conditions |
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What is a main characteristic of electron carriers?
|
They are extremely unstable by themselves
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What are electron carriers?
Examples of common ones? |
They store electrons in a form that can be later used to either generate ATP or synthesize new molecules
Common e- carriers are: NAD⁺, NADP⁺, FAD, & FMN |
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What is the difference between NAD⁺ and NADP⁺?
|
NAD⁺ is used for ATP production
NADP⁺ is used for biosynthesis |
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What is NAD?
Four things... |
1. Derived from niacin
2. “+” indicates nicotinamide ring which is in oxidized form 3. In reduced form its NADH (hydride transfer) 4. Both NAD⁺and NADP⁺ serve as mobile carriers (cofactors, one enzyme to another) |
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What are FAD and FMN?
Five things... |
1. Derived from riboflavin
2. Capable of 1 or 2 H transfers 3. FADH⁺ is semi-reduced 4. FADH₂ is fully reduced form 5. Both serve as tightly or covalently bound groups (prosthetic group) |
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|
What are dehydrogenases?
|
They are enzymes that use electron carriers such as NAD⁺, NADP⁺, FAD, or FMN
Specific dehydrogenases use specific electron carriers |
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|
What are vitamins?
Four facts... |
1. Vitamins are enzyme cofactors
2. Not fundamentally changed during enzyme reactions 3. Metabolic pathways are the major process that use vitamin cofactors 4. Humans must acquire vitamins through diet b/c we can't make them |
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What is niacin?
What does lack of niacin cause? |
Niacin is vitamin B3
It's found in yeast, liver, fish, and meat Lack of niacin causes: Black tongue (Pellagra) -Disease occurs in people whose diet is mainly corn and alcoholics -Symptoms include dermatitis, diarrhea, dementia, and death |
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What is riboflavin?
What does lack of riboflavin cause? |
Riboflavin is vitamin B2
-Found in milk, meats, and enriched grain products Lack of riboflavin causes: ariboflavinosis -Uncommon disease -Symptoms include dry mucus membranes of the mouth, eyes, and genitalia -Rarely fatal |
