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248 Cards in this Set

  • Front
  • Back
Why are membranes important?
1. Protection (barrier function and genetic material for replication), 2. Selectively Permeable (to create gradients), 3. Specialization (so different compartments can have different functions)
How are lipids divided into classes?
By structure, into fatty acids and steroids.
What are the primary lipids in biological membranes?
glycerophospholipids, sphingolipids, cholesterol
What is amphipathic?
Hydrophobic with a hydrophilic group
What is meant by lipids self-assemble in water?
due to the hydrophobic effect and van der Waals interactions
What is the most stable structure for a fatty acid?
Anti and extended
What is the symbol for a fatty acid?
[#C]:[# double bonds]
What is the common name and systematic name of this fatty acid? 18;)
common - stearic, systematic - octadecanoic
In what conformation do double bonds form?
cis conformation
What is the symbol, systematic name and common name of this structure?
18:1, 9-octadecenoic acid, oleic acid
What are PUFAs?
Poly-Unsaturated Fatty Acids
What is the symbol, systematic name and common name of this structure?
18:2, 9,12-octadecadienoic acid, linoleic acid
What are some factors that increase the melting point of fatty acids?
1. More C increases SA and energy gained by packing , 2. presence of double bonds
What has a lower melting point oleate, stearate or linoleate? Why?
Linoleate has a lower melting point because it has more double bonds although it has the same number of carbons.
Why are there essential fatty acids (EFA)? And what are they?
EFAs are required for normal health but cannot be made. This is because we cannot synthesize double bonds past C9.
What two EFAs do we need to obtain from our diet?
Omega-3 Fatty Acid (α-linoleic acid) and Omega-6 Fatty Acid (linoleic acid)
What is the naming convention for Omega Carbons?
They are labeled from the opposite direction (from the terminal c [ω end], the one farthest from the hydrophilic head, from the first carbon in the double bond)
What is an example of an Omega-3 fatty acid?
Fish oil.
What is α-linoleic acid converted to once in the body?
To elcosapentaenoic acid, which can be further converted to docosahexanoic acid
What is the systematic effecy of omega-3 fatty acids?
they are anti-inflammatory
What is an example of an omega-6 fatty acid?
vegetable oil
What is linoleic acid converted to once in the body?
arachidonic acid
What is the systematic effect of omega-6 fatty acids?
they are pro-inflammatory
What are the effects of unsaturated vs. saturated fatty acids on cardiovascular health?
Saturated fatty acids increase the risk of heart disease whereas unsaturated fatty acids decrease this risk.
Compare vegetable oil to elaidic acid, in structure, melting temperature, health benefits and commercial use?
Vegetable oil has two <b>cis</b>&nbsp;double bonds, Elaidic acid has one <b>trans</b>&nbsp;double bond. Vegetable oil melts at -9ºC, Elaidic acid melts at 45ºC. Vegetable oil is healthy but impractical for food industry due to short shelf life. Elaidic acid is unhealthy but great for baking and has a long shelf-life.
What is partial hydrogenation?
It is a reaction where there is a hydrogen limiting step, and results in one double bond converting from <b>cis</b>&nbsp;to <b>trans.</b>
What membrane fatty acids are attached to phasphate containing head groups?
1. glycerophospholipids and 2. sphingolipids
What is the most abundant membrane lipid in cells? And what does it’s structure depend on?
Glycerophospholipids are the most abundant membrane lipids, and they are based on the structure of glycerol attached to a single phosphate group.
What is conserved between glycerophospholipids and what is changed?
Glycerophospholipids ALWAYS have the same glycerol and phosphate structure. BUT, the fatty acid tails (length, degree of unsaturation) and head groups (size, charge) are variable to change.
What are the neutral glycerophospholipids?What are the neutral glycerophospholipids?
phosphatidylcholine and phosphatidylethanolamine
Why is phosphatidylcholine and phosphatidylethanolamine neutral?
Phosphate group (-1) and Head Group (+1) = 0
What are the zwitterionic fatty acids?
phosphatidylcholine and phosphatidylethanolamine
What are the anionic glycerophospholipids?
phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol (cardiolipin), phosphatidylinositol
What is the head group for diphosphatidylglycerol (cardiolipin)?
Subtracting the phosphate groups…it is the connection of two phospholipids bound by a glycerol
What determines which lipids are sphingolipids?
They are based on the structure of sphingosine.
What is the structure of the sphingosine backbone?
18 carbon amino alcohol backbone
How is sphingosine converted to ceramide?
By combining a sphingosine base and forming an amide bond to a fatty acid via ceramide synthase.
How is sphingomyelin formed?
Transfer of phosphocholine from phosphatidylcholine to a ceramide via sphingomyelin transferase.
What proportion of sphingolipids are sphingomyelin?
85%
What is the most common steroid in animals and the precursor for all other steroids?
cholesterol
Is cholesterol amphipathic? Why?
Yes, it has a small hydrophilic OH head group, but it is also rigid and plant.
What is the function of cholesterol in membranes, how does it accomplish this?
It modulates membrane fluidity by disrupting packing of hydrocarbon tails and slows the motion of hydrocarbon tails.
What is the lipid distribution of the mitochondrial membrane?
4% cholesterol, 17% cardiolipin, 5%phosphatidylinositol, 34%phosphatidylethanolamine, 38%phosphatidylcholine.
What is the lipid distribution in the plasma membrane?
28% cholesterol, 4%minor lipids, 6%phosphatidylserine, 6%phosphatidylinositol, 12%sphingolipids, 16%phosphatidylethanolamine, 28%phosphatidylcholine.
What are some sources of cholesterol?
1. synthesized by cells (mostly in liver) = 1000mg/day, 2. Diet (meats, cheese, butter, eggs) = 200-300mg/day
How is cholesterol transported in the body?
In lipoprotein particles
What is LDL?
low density lipoprotein, the low (protein:cholesterol) = low density. It brings cholesterol to tissues (and arteries). it is bad cholesterol since it promotes atherosclerosis.
What is HDL?
High density lipoprotein, High (protein:cholesterol) = high density. Carry cholesterol away from tissues, good cholesterol since it prevents atherosclerosis.
What was Akira Endo’s hypothesis on fungal and cholesterol?
Akira Endo hypothesized that fungi synthesize cholesterol inhibiting molecules to kill other microorganisms. So he tested over 6000 fungal species to find cholesterol synthesis inhibitors.
What do fungal membranes have in place of cholesterol?
ergosterol
What are statins? What was the first one Dr. Endo isolated?
Dr. Endo isolated a molecule from P. <i>citrinum</i>&nbsp;which he called mevastatin. It inhibited the HMB-CoA reductase (a cholesterol synthesis enzyme). Statins prevent coronary heart disease in humans, there are about 30 milion people taking statins and $35bilion in sales per year.
How do lipids self-assemble?
They spontaneously aggregate in water to bury their hydrophobic tails and expose their polar heads to water. The hydrophobic effect and van der Waals packing.
What does the final structure in water of a lipid depend on? What is the shape of fatty acids and glycerophospholipids?
The final structure in water depends on the shape of the individual lipid molecules. Fatty Acids are cone-shaped and Glycerophospholipids are cylinder-shaped.
What do cone-shaped lipids assemble into?
At low concentrations, the fatty acids will either be monomers or form monolayers. But, when they are above a critical concentration they will begin to form micelles.
How do cylinder-shaped lipids assemble in water?
They will spontaneously form lipid bilayers (e.g. phospholipids), either uni-lammelar vesicles (liposomes) or multi-lamellar vesicles (onion-like)
What does bilayer fluidity depend on?
THe lipid composition, the fatty acid tail saturation, the hydrocarbon chain length and the cholesterol content.
What is phase transition temperature (Tm)?
It is an index of membrane fluidity, the transition midpoint from gel-phase to fluid-phase.
What are the conformations of molecules in the gel and liquid crystal phases of the lipid bilayer? How does heat absorption change?
Heat absorption jumps at Tm, to return to normal levels when the structure has formed. This is the transition energy. In the gel phase, molecules are in the anti conformation whereas in the liquid crystal phase, they are in the gauche conformation.
How do lipid tails effect Tm?
Increasing size increases Tm (higher temp to transition). Increasing number of double bonds decreases Tm (lower temp to transition, easier due to less VDW and compactability).
How do head groups affect Tm?
Choline and Glycerol decrease Tm, Ethanolamine decreases much less.
How can phase transitions of the lipid bilayer be modulated?
The ‘sharpness’ will vary if it is a pure substance compared to a mosaic (it is broad for native membranes). Cholesterol will reduce packing at lower temperatures and reduce fluid motion at higher temperatures. And, cells will adjust lipid composition to maintain fluidity as environmental conditions change.
How do we measure lipid dynamics in membranes?
Technique: fluorescence recovery after photobleaching
How can we conduct fluorescence recovery after photobleaching?
<br>
How fast are lipids able to recover?
They can have rapid and complete recovery from bleaching, indicating that lipids diffuse rapidly in plane of membrane. A lipid can travel 2 μm in 1 second (cells are 1-100μm)
What is transverse diffusion? How are lipids able to accomplish this?
Transverse diffusion or lipid flip-flop is the conversion from the lipid being on one side to being on the other of the bilayer. Spontaneous flip-flop is very slow (taking hours to days) whereas membrane-embedded proteins may catalyze the flip-flop (taking seconds to minutes).
What are the proteins that can catalyze lipid flip-flop?
Flippase, Floppase and Scramblase (requires Ca2+). They need ATP to act.
How are membrane proteins distributed?
randomly
How do membrane proteins diffuse? How can we test this?
The test is injecting different coloured fluorescent markers and observing the pattern of diffusion. The conclusion is that membrane proteins diffuse laterally.
What is single molecule tracking of a phospholipid?
We label phosphatidylethanolamine with dioleoyl and add to liver kidney cell, then we track the label with microscope.
What is DOPE?
dioleyl-phosphatidylethanolamine
What are the results of the lipid tracking in the membrane experiment?
DOPE diffuses rapidly with compartments and ‘hops’ to adjacent compartments.
What suggests that membranes are not completely fluid?
That there are barriers to random diffusion since membrane proteins and lipids spend more time confined within compartments.
What are teh two types of membrane proteins?
Integral (intrinsic) and Peripheral (extrinsic)
How do globular proteins associate with membranes?
They can associate with them via non-covalent interactions (such as ionic and H-bond interactions, amphipathic α-helix, hydrophobic loop, association with integral protein). They are then peripheral proteins.
How can peripheral proteins be removed from membranes?
1. High salt - disrupts ionic interactions and hydrogen bonds by shielding charges, (aa-aa and aa-phospholipid interactions), 2. pH change - disrupts ionic interactions by protonating/deprotonating ionizable groups (Asp, Glu, His, Lys, Arg, Cys, Tyr)
What is an amphipathic α-helix, what are its two faces and what is it’s signature sequence?
It often has a polar-charged face and a non-polar face. The non-polar face inserts itself into the lipid bilayer. Its signature sequence is two polar, two non polar etc.
What is a requirement to be an integral protein?
to completely span the lipid bilayer.
How may we remove integral membrane proteins?
Detergents can displace native membrane lipids and form a micelle around hydrophobic regions, (i.e. detergent molecules compete for the lipid).
What do extracellular-facing regions of integral membrane proteins do?
They 1. receive signals from outside of cell via receptor sites and channel openings. 2. cell-to-cell communication and recognition via glycosylation on Ser/Thr (O-linked) and Asn (N-linked), it reads like a barcode.
What do the intracellular-facing regions of integral proteins do?
They transmit signals from outside, the short cytosolic ‘tails’ communicate to the inside by interacting with messenger proteins.
What does the membrane-spanning region of integral proteins function as?
1. the membrane spanning segments must interact with hydrophobi fatty acyl tails (in a hydrophobic environment, no water), 2. Amino acids in membrane spanning segments are mostly hydrophobic (80% are Leu, Ile, Val, Ala, Phe)
How can we identify membrane-spanning segments from a protein sequence?
membrane-spanning segments are rich in hydrophobic aa and are ~20-25 aa long, so we 1. give each aa a hydropathy score , 2. use a moving window procedure to find segments.
How do we develop a Hydropathy scale?
1. partition coefficients of aa: calculate fractional distributions between aq and organic phases, 2. Analyze known protein structures: calculate tendency of aa to be inferior (non-polar) vs. exterior (polar) on protein structures.
What is an amino acid partition coefficient (definition)?
It is the ratio of distribution of an aa between a hydrophobic solvent (n-octanol) and a hydrophilic solvent (water) at equilibrium. Since n-octanol and water are not miscible we are mimicking the lipid bilayer.
How do we calculate the aa partition coefficient?
e.g. of Ile
How do we measure hydropathy by analyzing protein structure?
Rationale: Polar aa will be found on water-exposed surface of protein, non-polar aa will be found on interior. W can vary very much on scales.
What do we define as a transmembrane segment, e.g. Glycophorin A?
Looking for stretches that are above 0 and have stretches of at least 20 amino acids. We look at the windows, which will dampen noise and really identify the regions we want to see.
Why is glycophorin A a prototypic membrane protein?
It is highyl glycosylated on ECM face, a 19-aa hydrophobic TM region, a short intracellular tail.
Why is the α-helix an ideal membrane spanning structure?
It has intramolecular H-bonds (i+4) that neutralize the polar backbone (by interacting with themselves they will not interact with the polar solvents, so they are effectively neutralized), The helix transverses membrane with minimal disrution of lipid packing. The hydrophobic side chains point out towards hydrophobic membrane.
How many integral proteins are single-spanning?
10-30%
What are some characteristics of single-spanning integral proteins?
They always cross the membrane as an α-helix. 20-25 residues are required to span 3-4 nm thick membrane.
How can we calculate the number of residues needed to span a membrane?
There is a 0.15 nm rise per aa, and there are 3.6 turns. We can calculate how many turns there are or how many aa based on length.
What is the hydrophobic helix paradox?
They are rich in β-sheet residues and contain very good helix breakers (e.g. Glycine). This is because backbone neutralization in membranes overcome aa structural propensities (CF rules are irrelevant to membrane proteins). And the mid-range Gly is involved in helix-helix interactions since it is small enough to allow them to approach each other.
How does Glycophorin A self associate via TM domains?
The two single-spanning proteins can come together as a dimer, they are non-covalently attached and Glycine will fall on one face of the helix and allow for very close packing.
Why membrane protein structure hard to analyze?
It is 1. difficult to isolate and purify (because hydrophobic), 2. requires detergents to solubilize (but when we add detergents to bind proteins they become much heavier and larger), 3. hard to crystallize (since in detergents they become squishy), 4. [Protein + detergent] = too big for NMR
What is the structure bacteriorhodopsin (bR)?
"<span style=""font-size: 107%;"">it is a multi-spanning membrane protein, that binds retinal (a chromophore). 7 α-helices perpendicular to membrane that surround a central retinal chromophore.</span>"
Why is bR such a good protein to study?
It was so abundant and pure in membranes that it formed hexagonal lattices in membranes.
Why does the purple color arise in bR? How is it bound to bR?
The color arises from a retinal molecule bound via a Schiff base linkage to Lys-216.
What is a Schiff base linkage?
Involve C=N, where N is bonded to an alkyl group in addition to H and is positively charged.
What does UV light do to retinal?
It causes a cis-trans isomerization between C13 and C14.
How does the cis-trans isomerization of retinal enable a series of proton transfers?
1. NH+ on retinal protonates Asp 85. 2. Asp 85 releases &nbsp;H+ extracellularly, 3. Asp 96 protonates N on retinal, 4. H+ reserves in the cell protonate Asp 96.
How does bR allow cells to create ATP?
The pumping of H+ extracellularly creates a concentration gradient. This proton gradient is used via ATP synthase, which brings protons back into the cell and creates ATP.
What diversions of multi-span helical membrane structures allow for movement and structural changes?
Long-tilted helices, re-entrant loops, and non-helical segments
When can integral protein form β-sheets?
When the N- and C-terminal strands H-bond with each other to form a cylindrical β-barrel. We need at least 8 strands.
What TM structures do porins have and where are they found?
They have β-barrels, are found in gram-negative bacteria (E.coli), mitochondri and chloroplasts, and allow small molecules ot pass (mono-, disaccharides and phosphate).
What is the sequence motif for a β-barrel? Why?
This allows for the interior polar residues to face the hydrophilic portion and the exterior non-polar residues to face hydrophobic membrane.
How do pathogenic bacteria use β-barrels? E.g. hemolysin
They construct them via different subunits, where soluble monomers assemble on the membrane and form β-barrel pores which cause cells to leak and die. E.g. hemolysin forms a heptomeric hemolysin pore, a 14-stranded β-barrel (made from 7 monomers) which cause blood cells to lyse.
What categories of molecules can passively diffuse and what categories need facilitated diffusion?
Gases and small uncharged molecule (e.g. ethanol) can passively diffuse. Water is slow to diffuse, but can do so more quickly when facilitated. Large uncharged, ions and charged polar need to be facilitated.
How does the rate of transport differ between increasing concentrations of substrate?
Facilitated diffusion proceeds very fast at low concentrations but then becomes saturated and experiences diminishin returns. Passive diffusion experiences almost direct linear realtion.
What are some hallmark features of ion channels/
1. Selectivity, 2. Conduct ions at very high rates (10^8 per second), 3. Gate (open and closes, not continuous channel)
What is the potassium ion channel essential for?
regulation of cell volume, secretion of hormones, electrical impulse formation
What is the structure of a K+ channel?
It is composed of four identical subunits coming together
What is the selectivity filter?
"Each subunit of the K+ tetramer contributes a five aa reentrant loop (TVGYG) into the channel lumen (this is the selectivity filter). There are 4 backbone carbonyls + Thr side-chain OH. This creates four possible binding sites for K+ ions, where it will be surrounded by 8 O<span style=""font-size: 107%;"">&nbsp;and mimick the structure of a hydrated K+. There can be 1-4 K+ bound.</span>"
Why are K+ channels ion selective?
The filters are fine tuned to accomodate a K+ ion. It cannot shrink to bind smaller Na+ ions. Hence there is a &nbsp;higher energetic cost for Na+ ions (easier stabilized in water). Therefore selectivity is achieved.
How is potassium transported in the K+ channel?
There are only two K+ in the filter at any one time, either at positions 1 and 3 or 2 and 4. Water occupies the other sites.
When does the K+ channel close?
In response to intracellular pH, helix bending at a conserved Gly residue occurs. Gly acts as a molecular hinge to gate open/closed.
What process do membrane receptors mediate?
1. sense an external stimulus (i.e. hormone), 2. transduce to secondary messengers, 3. trigger a cellular response.
What is the structure of G proteins?
They are hetero-trimers. α and γ are lipid anchored, α binds to GDP in the inactivated state.
What does agonist binding of GPCR do?
It activates Gα (the transducer), which rapidly exchanges GDP to GTP. Gα-GTP dissociates from Gβγ and Gα bind effector enzyme (diffuses through membrane laterally). Can activate PLC or AC.
How do we turn off a GPCR-AC mediated signal?
1. Gα has intrinsic GTPase activity (GTP -&gt; GDP) where Gα (GDP) dissociates from AC, 2. cAMP is quickly degraded after synthesis by phosphodiesterase (to 5’ AMP)
How does ligand binding affect the β2-adrenergic receptor?
It induces very small changes locally on the extracellular face, but there are large conformational changes inside by TM α-helix 6.
How does the conformation change of β2-adrenergic receptor binding lead to GDP release of Gα?
The small movment of the protein causes large movement of Gα, and this promotes GDP release.
What are properties of enzymes?
1) They are highly specific for their reactants or substrates (S), 2) Many exhibit sterospecificity, 3) Specificity of the reaction, 4) Enzyme (E) means ‘present in cells’, 5) Sumner crystallized urease in 1926, 5) Almost all enzymes are proteins, 6) Classification, nomenclature, kinetics, regulation of E activity coenzymes (required by E for full activity)
What does it mean when you say enzymes exhibit stereospecifity?
That they prefer L isomer over D isomer
What is urease?
It is a bacteria enzyme that uses urea to carry out its reactions.
What are the 6 classes of enzymes?
1. oxidoreductases (oxidation-reduction reactions), 2. transferases (transfer of functional groups), 3. Hydrolases (hydrolysis reaction), 4. Lyases (addition to double bonds), 5. Isomerases (isomerization reactions), 6. Ligases (formation of bonds with ATP cleavage)
What enzymes are oxidoreductases?
enzymes called oxidases, dehydrogenases, reductases
What type of reactions do oxidoreductases undergo?
Oxidation-Reduction reactions where one substrate is oxidized (loss of electrons) and one is reduced (gain of electrons)
What does NAD+ stand for?
nicotinamide adenine dinucleotide
What enzyme converts L-lactate + NAD+ -&gt; pyruvate + NADH + H+?
lactate dehydrogenase
What type of reaction do transferases perform?
group transfer reactions (using ATP). E.g. kinases carry out phosphorylation.
What is the enzyme that converts L-alanine + α-ketoglutarate -&gt; pyruvate + L-glutamate? What type of reaction is this?
It is a transferase called alanine transaminase
What reaction does carnitine acyltransferase mediate?
It transfers an acyl group to 1-carnitine from fatty acyl CoA (fatty acid in activated form attached to CoA). This creates an acylcarnitine and CoA.
What type of reaction do hydrolases perform?
Catalyse hydrolysis
What enzymes are hydrolases?
proteases, lipases, nucleases, esterases
What reaction does pyrophosphatase mediate?
Pyrophosphatase uses water to break PPi (pyrophosphate). This reaction yields 2 phosphates = hydrolysis of anhydride bond (that links phosphates)
What enzyme mediates the conversion of pyrophosphate to 2 phosphate?
pyrophosphatase.
What type of reaction do lyases mediate?
They create a double bond or addition to a double bond
What enzymes are lyases?
synthases since they catalyze addition reactions
What enzyme mediates the conversion of pyruvate + H+ -&gt; acetaldehyde + CO2?
pyruvate decarboxylase
What reaction does fatty acid synthase mediate?
The creation of fatty acids and release of CO2 (to provide energy)
What type of reaction do isomerases mediate?
They catalyze an isomerization reaction
What enzyme converts 1,3-Biphosphoglycerate to 2,3-Biphosphoglycerate?
Biphosphoglycerate Mutase
What type of reaction do ligases perform?
catalyze the joining of two molecules or ligation
What are two products of a ligation reaction that are always there?
ADP and phosphate (Pi)
What enzyme combine L-glutamate + NH4 to create L-glutamine?
glutamine synthetase
What reaction does Fatty acyl synthetase mediate?
ATP drives the formation of link between CoA and a fatty acid
What is the catalytic power of an enzyme defined as?
the ratio of enzyme catalyzed rate of a reaction to the uncatalyzed rate.
What are coenzymes and cofactors?
They are non-protein components essential to enzyme activity.
How much may enzymes accelerate reactions?
Up to 10^16 over uncatalyzed rates.
How much products do enzymes yield?
&gt;95%
How is specificity of an enzyme controlled?
By the structure, the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield.
What type of enzyme is urease? What does it use as a substrate? What is its general reaction?
It is a hydrolase, that only uses urea as a substrate. It breaks it down into ammonia and carbon dioxide.
What type of reaction does alcohol dehydrogenase mediate? What is its general reaction? What is its substrate?
It is an oxidoreductase reaction, that only uses alcohols.
What reaction does hexokinase mediate? What is its substrate? What is its general reaction?
It is a transferase, that uses glucose and ATP as substrate (not energy) to yield ADP and g-6-p
What reactions do proteases mediate? What is its substrate? What is its general reaction?
It is a hydrolase reaction, it uses a peptide bond as its substrate.
What reactions do esterases mediate? What is its substrate? What is its general reaction?
It is a hydrolase, ester bonds are its substrate.
Where does trypsin cleave?
Only after basic residues.
What are 6 properties of active sites?
<div>1. specialized regions within enzyme necessary for substrate binding and catalysis</div><div>2. typically tend to be 3D clefts or crevices, formed from distant regions of polypeptide chain are brought together</div><div>3. have a unique microenvironment, typically exclude water (except hydrolases)</div><div>4. usually substrates bind within active sites by non-covalent interactions (hydrogen bonds,van der waals, ionic charge interactions, etc.)</div><div>5. tend to have 2-3 key residues needed for catalysis</div><div>6. active site make up a very small percentage (&lt;1%) of enzyme structure/volume (why? other binding sites for regulation)</div>
What is the function of lysozyme?
It lyses bacterial cell walls, the active site can hold up to 6 sugars from the wall at any time.
What is the structure of lysozyme?
It is approximately 120 residues in length. It has a cleft, and Glu35 and Asp52 are the key residues.
What is the composition of the sugars in bacterial cell walls?
N-Acetyl Glucosamine (GlcNAc) and N-Acetyl Muramic Acid (MurNAc)
How are enzymes regulated?
1.substrate availability, 2. covalent modification, 3. allosteric control, 4. zymogens, proenzymes
What is the injury pathway leading to conversion of Arachidonic Acid to Eicosanoids?
It is the wounding response, when a phospholipid in the membrane is injured (e.g. heart attack or stroke), the enzyme PLA2 becomes activated. The acyl chain at position 2 of the degraded phospholipid is cleaved to become polyunsaturated (20:4), Omega-6 Arachidonic Acid. Cyclooxygenase adds O2 to AA to become an eicosanoids which elicits thrombus formation, vasoconstriction, pain and fever.
What is the enzyme that cleaves a degraded phospholipid in the wounded resposne?
PLA2
What is an example of covalent modification of enzymes?
Phosphorylation, attach a phosphate from ATP to an enzyme. Residues typically phosphorylated are Ser, Tyr and Thr (all -OH).
How does covalent modification regulate pyruvate dehydrogenase activity?
In the phosphorylated state, via pyruvate dehydrogenase kinase (transferase) it is inactive - other product is ADP. In the un-phosphorylated state, via pyruvate dehydrogenase phosphorylase (hydrolase) it is active - use H2O to yield Pi as product.
How is glycogen covalently modified to Glucose-1-Phosphate?
Phosphorylase b Kinase activates Phosphorylase b (inactive) to Phosphorylase a (active). Phosphorylase a performs phosphorylysis with glycogen as a substrate - cleaves a glycosidic bond and simultaneously adds phosphate at the 1-position.
Where are allosteric sites located?
Either directly on an enzyme or on the regultory subunit if it is a polymer.
How is allosteric control regulated a useful feedback mechanism?
for example, in the conversion of Thr to Ile. E1 (Thr deaminase) begins the pathway of numerous steps leading to the synthesis of Ile. When there is enough Ile present, it binds to allosteric site on Thr deaminase to halt production.
What is phosphofructosekinase-1?
It is an enzyme in the glycolysis pathway that converts fructose-6-phosphate to fructose-1,6-phosphate
How is energy (ATP production) regulated via allosteric control?
When there is too much ATP, then there is little activation of PFK-1 and so there is little production of pyruvate and ATP. When there is too little ATP, meaning that there is a lot of ADP. ADP binds to PFK-1 and activates it to increase energy production.
How does loss of allosteric control lead to the disease state of ‘gout’?
It is the irregular negative feedback of phosphorribosylpyrophosphate synthetase (PRS). This causes increased levels of urate/uric acid which accumulates in the lining and fluid of joints in hands, knees and feet. Leads to inflammation and pain.<div><br></div>
How is enzymatic activity regulated by zymogens and proenzymes?
They are inactive enzyme precursors, and they become activated as needed.
How does cleavage of trypsinogen by enteropeptidase lead to activated trypsin?
Trypsinogen is found in the duodenum, and when digesting enteropeptidase cleave 6 N-terminal amino acids from trypsinogen to create activated trypsin. Trypsin can positively feedback to activate more trypsin. Trypsin can further activate digestive enzymes chymotrypsinogen and proelastase to chymotrypsin and elastase respectively.
Where are chymotrypsinogen and proelastase synthesized?
in the pancreas
What is Beer’s law and what does each letter denote?
A = absorbance, ε = molar absorptivity, l = the distance the light travels through the material (i.e. path length), c = molar concentation
What is an enzymatic assay?
We take a particular enzyme and add to substrate, see if a particular product can be formed.
What is the formula for enzymatic rate?
mM/min
What is the rate constant?
time^-1
What is the rate constant?
It is the slope of the graph where [S] is plotted on the X-axis and Vo is plotted on the Y-axis.v=k[s]
Rhe Michaelis-Menten Equation? What does it assume?
Assumes: 1. the formation of an enzyme-substrate complex, 2. that the ES complex is in rapid equilibrium with free enzymes, 3. breakdown of ES to form products is assumed to be slower than formation of ES and breakdown of ES to re-form E and S.
What is the Michaelis-Menten rates?
1) Rate of P production: v=K2[ES], 2) Rate of ES formation: k1[E][S], 3) Rate of ES breakdown: (k-1 + k2) [ES], Steady State: k1[E][S] = (k-1 + k2)[ES].
When [S] = Km what is V?
V = 1/2Vmax
What is Km?
It is the kinetic activator constant. It is derived from rate constants, is only true under Michaelis-Menten conditions. Small Km means tight binding, high Km means weak binding.
What is Vmax?
It is the theoretical maximal velocity. It is constant, It is never achieved in reality. To reach it, we would need all enzyme molecules to be tightly bound with substrate. Vmax is asymptotically approached as substrate is increased.
What is kcat?
the turnover number, the number of substrate molecules &nbsp;converted to product per enzyme molecule per unit of time, when E is saturated with substrate
What is kcat equal to?
It can range from &nbsp;less than 1 sec to many millions &nbsp;per second.
What are the effects of inhibitors on different types of inhibition?
Apparent Km is increased in competitive inhibition since I binds to E only. Apparent Vmax decreases since I binds to E or ES.
How is succinate dehydrogenase an example of classic inhibition?
The typical substrate is succinate, but the competitive inhibitor is malonate.
Why is Vmax decreasing but Km stays the same in non-competitive inhibition?
Because we cannot go forward to produce the product .
What is the slope in an inversed M-M equation graph?
Km/Vmax
What is irreversible enzyme inhibition? Explain using aspirin and Cox.
In this situation, the inhibitor changes the enzyme and causes it to be inactive, the only way to recover from it is to synthesize more enzyme. Aspirin attaches to Ser533 in the active site. This destroys the enzyme because it will not unbind, and Ser533 is critical in the active site.
What is the function of glycopeptide transpeptidase?
It makes bacterial cell walls.
How does penicillin work?
The reactive peptide bond of β-lactam ring binds to OH of Ser on Glycopeptide transpeptidase creating an penicilloyl-enzyme complex (enzymatically inactive). This halts synthesis of bacterial cell walls and thus cell growth.
What five aa account for 66% of all catalytic residues that are charged?
His, Asp, Arg, Glu, Lys
What is the structure of lysozyme’s active site?
It has 7 pockets which accommodate a single GlcNAc or MurNAc. Residues ABCEF are in the chair conformation but residue D must go into half-chair confomration of MurNAc.
What is occurs at the D-E glycosidic linkage site when bound to a lysozyme?
Glu-35 (protonated at pH 5) acts as an acid catalyst, which donates a proton to the O in the glycosidic linkage which severs the D-E linkage. E-F leaves the active site.
What occurs at the E-F site in the lysozyme after the cleavage of D-E?
The portion of the substrate bound in sites E and F(an alcohol leaving group) diffuse out of the cleftand is replaced by a molecule of water.
What occurs after H20 replaces E,F pockets in the lysozyme active site?
There is a partial double bond between the D terminal Carbon and its adjacent O. It is now an oxocarbocation. The Asp-52 residue (negatively charged at pH 5) forms an ion pair with it, which resembles a covalent bond (but it isn’t)!
What happens after Asp-52 forms a ionic interaction with D in a lysozyme?
Glu-35 receives a proton from the H2O in E. The OH- ion adds to the oxocarbocation. This creates a new hydroxyl group and then A-D diffuses out of the active site.
What are the steps of the acid-base catalytic cleave of sugars via a lysozyme?
1. Sugar binds into pockets A-F, 2. Glycosidic linkage between D-E is cleaved, proton is taken from Glu-35. 3. E-F diffuses out of the cell and is replaced by H2O, 4. Carbon on D that was involved in glycosidic linkage forms oxocarbocation with adjacent oxygen. 5. Asp-52 forms stabilizing bond with oxocarbocation. 6. Glu-35 attacks and takes proton from H20 leaving OH-, 7. OH- attacks carbocation and now is stable. 8. A-D diffuse out of the cell.
Where does subtilisin cleave a peptide bond on Ribonuclease A (RNase A)?
betwee residues 20 and 21.
When subtilisin cleave RNase A, what is the end product?
RNase S
Why is RNase S active, but not S peptide or S protein?
It is because both are needed to create an active site. RNase has strong enough non-covalent interactions that the molecule still has activity,
How many H are found in the S peptide? S protein?
12 in the S peptide and 119 in S protein
How can we test if His 12 and His 119 are involved in the active site of RNase?
We can chemically modify them. We can use an agent such as Iodoacetate, and incubate them. The acetate group will bind to the electrons on the imidazole ring. As it becomes carboxylmethyl Histidine and its transfer of the proton is blocked.
What does RNase A cleave?
It cleaves at pyrimidines sites (so cytosine and thymine in RNA) on the 3’ side.
What is the structure of the &nbsp;pyrimidine binding pocket in RNase? What is the mechanism of action?
His-12 is deprotonated, His-119 is protonated. Lys -41 plays a role in stabilizing transition states. H-12 takes proton from 5’ ribose and becomes protonated. 2. H-119 donates proton to phosphate between sugars. 3. transition bonds form between H-119 imidazole and phosphate group OH, and between NH3+ on Lys-41 and O- on phosphate group. 4. 2nd molecule’s 5’ phosphodiester bond is cleaved where it binds to OH on phosphate. Forms new sugar. Leads to intermediate State 1., 5. H2O comes in and His-119 binds H, OH- attacks P in phosphate creating P bound to 2 O, 2O-, 1 OH. NH3+ on Lys-41 stabilizes one O-, 6. One O takes H from imidazole of H-12, and this leaves only 4 O’s bound to P. 7. Double bond forms between 1 O- and no longer needs Lys-41. 8. Product is 3’- Nucleosidemonophosphate.
What is step 1 of the mechanism of RNA cleavage by RNase A?
H-12 binds H of 2’ OH. Leaves O to bind P. N-H of His-119 binds O- of phosphate.
What is step 2 of the mechanism of RNA cleavage by RNase A?
The unstable transition states have O from phosphoester linkage (on 5’ carbon of second nucleotide) bind H of adjacet OH (on phosphate). An O- stabilized by Lys-41 forms a double bond to compensate.
What is step 3 of RNA cleavage mechanism by RNase A?
H20 comes in. N of H-119 bind H, forming OH-. OH- attacks P. P=O become P-O-.
What is Step 4 of RNA cleavage mechanism by RNase A?
P-O bond bound to 2’ Carbon (used to be O-H), is regenerated since it takes H from H-12. P-O- reforms double bond to be P=O.
What are the serine proteases?
chymotrypsin, trypsin and elastase
What are some characteristics of the active site of serine proteases?
"They form a catalytic triad between H-57, S-195 and D-102. <span style=""font-size: 114%;"">The imidazole ring of His-57 removes the
What is Step 1 of the mechanism of chymotrypsin catalyzed cleavage?
Imidazole attaches to proton of OH on Ser 195. Ser 195 forms bond to C of peptide bond after aromatic residue. C=O becomes C-O-. It becomes a tetrahedral-like from trigonal-like shape.
What is Step 2 of the mechanism of chymotrypsin catalyzed cleavage?
NH on aa after peptide bond attaches to proton of N-H on imidazole ring.
What is Step 3 of the mechanism of chymotrypsin catalyzed cleavage?
C-O- becomes C=O as NH2-R is cleaved.
What is Step 4 of the mechanism of chymotrypsin catalyzed cleavage?
Amine Product (P1) leaves and H2O enters.
What is Step 5 of the mechanism of chymotrypsin catalyzed cleavage?
H2O donates proton to N of imidazole ring. OH- attacks C=O to become C-O-.
What is Step 6 of the mechanism of chymotrypsin catalyzed cleavage?
His 57 reforms, donated proton to Ser 195 which breaks up bond with peptide. C-O- reforms C=O, returns to trigonal structure.
What is Step 7 of the mechanism of chymotrypsin catalyzed cleavage?
Dissociates under hydrophobic conditions with a new carboxyl product (P2)
How is the transition state stabilized in chymotrypsin?
The TS involves two tetrahedral oxyanion intermediates. They are stabilized by a pair of amide groups called the oxyanion hole.
What can cofactors be broken up into?
essential ions and coenzymes
What essential ions are cofactors?
"There are loosely bound activator ions and tightly bound metal ions of metalloenzymes. Mainly Mg<span style=""font-size: 107%;"">2+, Ca2+, Fe2+, Co+.</span>"
What groups of organic coenzymes are there?
Cosubstrates (loosely bound), Prosthetic groups (tightly bound).
What is the structure of NAD+ or NADP+ or NADH or NADPH, what is it composed of?
it is composed of two groups joined by a diphosphoester linkage. 1. nicotinamide mononucleotide (NMN) 2. adenosine monophosphate (AMP)
What is the difference between the Oxidized and Reduced form of NAD+/NADH?
Nicotinamide group has one less H in oxidized (NAD+) which leads to a positively charged ring. In the reduced form, it is neutral and there are 2 H.
What is FAD?
It attaches to enzymes that catalyze reactions. It becomes reduced to FADH2. E.g. E-FAD (where E is succinate dehydrogenase SDH) is involved in the breaking up of succinate.
What is riboflavin, or Vitamin B2?
Minus the blue phosphate group
What is FAD?
Flavin Adenosine Dinucleotide.
What is a phosphanhydride linkage?
Bond between two adjacent phosphate groups.
What are the differences between Mb and Hb?
Hb transports O2, Mb stores O2. Mb is monomeric whereas Hb is tetrameric. Mb’s single peptide is 153 aa long, 17,200 Da. Hb has two α chains of 141 residues and 2 β chains of 146 residues.
What is the structure of Protoporphyrin IX?
There are 4 pyrrole rings. All linked together via methene bridges (C=CH).
What is the structure of heme or Fe-protoporphryin IX?
The N of each pyrrole ring coordinates bonds around the Fe central atom. In Fe2+ state it is ferrous and in Fe3+ it is ferric.
When does heme bind to O2?
When it is bound in the ferric state. The Fe3+ bound is called metmyoglobin or methemoglobin because it does not bind O2.
What is the composition of Hb in red blood cells?
They are 90% of the intracellular protein and there is approx 300 billion in each.
What make up the fifth and sixth coordinate bonds binding heme? When does this occur?
The fifth bond occurs with the proximal histidine (His-93). But the sixth bond can only occur when there is O2 bound, and it happens with the distal histidine (His-64)
Why is binding reversible in heme groups?
Because Val 68 and Phe 43 are very hydrophobic, they form a hydrophobic cleft which makes the binding reversible. This is because when left in hydrophilic environment Fe2+ is permanently converted to Fe3+, only the partial transferration in hydrophobic pocket allows reversal.
What is the P50 of Mb compared to Hb? What does P50 mean?
It is the concentration where 50 % of the enzyme is bound to its subtrate. Mb = 2.8 and Hb = 26. This is because Mb has a greater affinity for O2 since it stores it whereas Hb needs to release it into tissue.
What does the sigmoidal curve of Hb suggest?
It suggests that there is cooperativity. When one unit of Hb binds O2, this facilitates the other subunits to bind O2.
What are the two states of Hb and what do their graphs look like?
The R-state is the high affinity state, which has a hyperbolic curve (like Mb) and likes to bind O2. The T-state is the low affinity state and doesn’t like to bind O2. They are in equilibrium and when we combine the two scores we achieve the sigmoidal Hb curve.
How does binding of O2 to Fe2+ in one subunit induce the conformational change that favors other subunits binding?
Deoxygenated Hb has Fe2+ out of the heme plane by 0.06nm, but when it is oxygenated the atom moves 0.049nm closer to the plane. This drags His and the F-helix with it. The change transmitted to the subunit interfaces, lead to the rupture of salt bridges.
How is 2,3-BPG an allosteric modulator/effector of Hb?
It is found at high concentrations in red blood cells, and has 5 negative charges. It will bind at a different site on Hb to regulate binding to Hb. It binds inside the central cavity because the negative charges like interacting with the positive charges (2 Lys, 4 His, 2 N-termini) on the interface of the Hb active site. It favors and stabilizes the T-state (opposite of O2 which stabilizes the R-state).
Why does Fetal Hb have a lower affinity for 2,3-BPG?
It needs to retain O2 that it obtains from its mother.
Why does H+ stabilize the T-state of Hb?
"When we decrease pH (increase [H+]), such as in cells that are metabolizing really quickly to produce CO2 -&gt; H2CO3 (carbonic acid) -&gt; H+ + HCO3-. Deoxy-Hb has a greater affinity for O2 than oxy-Hb, and so dissociation of O2 is enhanced and is prevented from being reoxygenated. HbO2 + H+ -&gt; HbH+ + O2<div class=""page"" title=""Page 22"">
What is the Bohr effect?
It is the effect of H+ on O2, where binding or protons diminishes oxygen binding and binding of oxygen diminishes proton binding.
How does CO2 stabilize the T-state of Hb?
Some CO2 is bound to Hb and transported in the form of &nbsp;carbamate (NHCOO). When it is released it can become oxygenated but otherwise it isn’t.