Ch 10, 11, 13, 14, 16 Flashcards
Nucleic Acids, Fats, Glycolysis, CTA
Calculate the standard free-energy change of the reaction catalyzed by the enzyme phosphoglucomutase given that starting with 20 mM glucose 1-phosphate and no glucose 6-phosphate, the final equilibrium mixture at 25ºC and pH 7.0 contains 1.0 mM glucose 1-phosphate and 19 mM glucose 6-phosphate.
Does the reaction in the direction of glucose 6-phosphate formation proceed with a loss or a gain of free energy?
First, calculate the equilibrium constant.
Then, calculate the standard free-energy change
What is the difference between ∆Gº’ and ∆G?
How are they related?
The standard transformed free energy change (∆Gº’) tells us in which direction and how far a given reaction must go to reach equilibrium when the concentration of each component is 1.0 M, the pH is 7.0, the temperature is 25ºC, and the pressure is 101.3 kPa (1 atm). Thus ∆Gº’ is a constant.
The actual free energy change/Gibbs free energy (∆G) is a function of reactant and product concentrations and of the temperature prevailing during the reaction. The ∆G of any reaction proceeding spontaneously toward its equilibrium is always negative, becomes less negative as the reaction proceeds, and is zero at the point of equilibrium.
actual free-energy change (∆G) = 0 vs standard free-energy change (∆Gº’) = 0
∆G=0: implies that the reaction is at equilibrium (no more work is done by the reaction in either direction)
∆Gº’ = 0: implies that the equilibrium constant (the concentrations of reactants and products at equilibrium) is 1
What are the conditions in which metabolic reactions with a large positive ∆Gº’ can still be exergonic?
A reaction with a positive standard-free energy change can go in the forward direction if ∆G is negative.
Immediate removal of products to keep the ratio of products to reactants well below 1 so that the term RTlnQ has a large, negative value
FRAP
Fluorescence Recovery After Photobleaching
allows us to monitor lateral lipid diffusion by monitoring the rate of fluorescence return; the diffusion coefficient of lipid in the leaflet can be determined
rates of lateral diffusion are high (up to 1µm/sec); a lipid can circumnavigate E. coli cell in one second
Membrane Rafts
there’s also different lipid distribution within a membrane (not only between membranes)
lipid rafts
- contain clusters of glycosphingolipids with longer-than-usual tails (more opportunity for van der Waals interaction, giving more stability)
- are more ordered
- contain specific doubly or triply acylated proteins
- allow segregation of proteins in the membrane
Caveolin
when several caveolin dimers are concentrated in a small region (a raft) they force a curvature in the lipid bilayer, forming a caveola
flattening of caveolae allows the plasma membrane to expand (increase surface area)
other modes (besides caveolin) of membrane curvature
- a protein with intrinsic curvature and a high density of positive charge on its concave surface
- a protein with one or several amphipathic helices inserted into the outer leaflet of the bilayer
- proteins with BAR domains can polymerize into a superstructure that favors and maintains the curvature
membrane fusion
membranes can fuse with each other without losing continuity
fusion can be spontaneous or protein-mediated
examples of protein-mediated fusion: entry of influenza virus into the host cell and release of neurotransmitters at nerve synapses
Why is passive diffusion of polar molecules across membranes unfavorable?
involves desolvation and thus has a high activation barrier
What are the 3 classes of transport systems?
- uniport: facilitated diffusion
- symport (cotransport): secondary active transport
- antiport (cotransport): secondary active transport
Glucose transport
- glucose in blood plasma binds to T1
- conformational change from T1 to T2
- glucose is released from T2 into the cytoplasm
- the transporter returns to T1
There are multiple glucose transporters
A Na+-glucose symporter (intestinal lumen) and a glucose uniporter (blood) operate on opposite sides of epithelial cells
cells can also have asymmetry with distinct proteins confined to one side
Bicarbonate transporter
antiporter: chloride-bicarbonate exchanger
maintains the electrochemical potential across the membrane
two types of active transport
a) primary active transport: energy released by ATP hydrolysis drives solute movement against an electrochemical gradient
b) secondary active transport: a gradient of an ion has been established by primary active transport; movement of S1 down its gradient now provides energy to drive cotransport of a second solute against its electrochemical gradient
ABC Transporters
a large family of ATP-driven transporters that pump ions and molecules against a concentration gradient
located in the plasma membrane, in the ER, mitochondria, and lysosomes
CFTR protein of the plasma membrane (airway epithelium):
- ion channel for Cl
- when it doesn’t work, cystic fibrosis; mucus accumulates, entrapping bacteria that results in infection of lungs and airway
Proton pumps
ATPase uses ATP to pump protons through the membrane
the energy of the proton gradient can be used to make ATP (in chloroplast and mitochondrial membranes) by ATP synthase
aquaporins
allow rapid water passage through membranes
size restriction: the pore narrows at His 180 to a certain diameter, limiting the passage of molecules larger than H2O.
electrostatic repulsion: the positive charge of Arg 195 repels cations, including H3O+
water dipole reorientation: the helices with Asn-Pro-Ala are oriented with their positively charged dipoles pointed at the pore to force H2O to reorient as it passes through; this breaks up hydrogen-bonded chains of water molecules and prevents proton passage by proton hopping
K+ channels and their specificity
at the inner and outer plasma membrane surfaces, the entryways to the channel have several negatively charged amino acid residues
the pore is a specific diameter that allows a specific ion and its water shell to pass through the channel
How do major nucleotide mutations occur?
naturally
radiation
reactive chemicals
Accumulation of nucleotide mutations is linked to what?
aging and carcinogenesis
What are the two types of naturally occurring mutation reactions?
deamination and depurination
deamination
very slow reaction; occurs one in 107 cytosine residues per day (~100 spontaneous events per day); adenine to guanine occurs much slower (1/100th the rate of C to U)
cytosine is mutated to uracil; uracil is recognized as foreign in DNA and removed by a repair system
thymine allows for long-term storage of genetic information; otherwise, AU not recognized as mutation, and eventually GC will be eliminated
depurination
N-glycosidic bond is hydrolyzed; the base is lost, creating an abasic site
purines are lost at a higher rate than pyrimidines (10,000 purines lost per day in one mammalian cell); depurination of RNA is much slower and less physiologically significant
proteins recognize damage and fill in gap
What are the two types of mutations caused by radiation?
- thymine dimers (UV)
- cause kink in DNA, blocking the movement of polymerases
- fixed by nucleotide excision repair (E. coli handles the repair differently than humans)
- fragmentation of nucleic acids (ionizing radiation like x rays and gamma rays)
- difficult to fix
- high energy traverses the cell and collides with water, resulting in oxygen radicals that react with molecules in the cell
What are the two different ways reactive chemicals can cause nucleotide mutations?
- oxidative damage
- reactive oxygen species arise during irradiation or as a byproduct of aerobic metabolism (mitochondrial DNA is most susceptible)
- damage DNA: oxidation of deoxyribose, hydroxylation of guanine, and break strands; accumulate as you age
- cell destroy reactive oxygen species, but a fraction escape
- alkylating agents
- dimethylsulfate can methylate guanine, preventing base pair with C
- S-adenosyl methionine normally present in cells (not a mutagen)
- cancer treatment: lots of side effects because we’re killing good cells too
What are some other functions of nucleotides besides the storage of genetic information?
- energy source: ATP, UTP, GTP, CTP
- hydrolysis of ester linkage and anhydride bond in nucleoside triphosphates provides chemical energy to drive many reactions
- components of enzyme cofactors: adenine dinucleotides (Coenzyme A, NAD+, FAD)
- adenosine do not directly participate in the primary function, but the removal of adenosine results in a drastic reduction of cofactor activities
- regulatory molecules: second messengers bind to receptors on the cell surface, leading to changes in the cell interior
- cAMP: slime molds, unicellular form, and fruiting body
- ppGpp: produced in bacteria in response to amino acid starvation; inhibits synthesis of tRNA and rRNA
- cGMP
DNA Sequencing
- makes a DNA strand complementary to the strand under analysis
- a primer is annealed to the template DNA and polymerase extends it
- ddNTPs are added in small amounts (10%) along with lots of dNTPs
- ddNTPs interrupt DNA synthesis because they lack the 3’ –OH needed to add the next nucleotide
- a small fraction of the synthesized strands are prematurely terminated, resulting in a solution with a mixture of fragments
- the different-sized fragments are separated by electrophoresis; the shortest strands (sequences closest to the primer) move farthest
- now reading is automated: each of the ddNTPs is fluorescently tagged with a different color; expose to UV light and the computer helps with sequencing
What are the different types of lipids and their classes?
- lipids that do not contain fatty acids: cholesterol, terpenes
- lipids that contain fatty acids (complex lipids)
- storage lipids (hydrophobic)
- triacylglycerols
- wax
- membrane lipids (hydrophilic; charged head groups)
- glycerophospholipids
- sphingolipids
- storage lipids (hydrophobic)
Fatty Acids
carboxylic acids with hydrocarbon chains containing 4 to 36 carbon atoms
almost all naturally occurring fatty acids (in our diets) have an even number of carbons in an unbranched chain of 12 to 24
hydrocarbon chains are saturated, monounsaturated (oleic acid), or polyunsaturated (omega-3 fatty acids: ALA, DHA, EPA)
humans can’t make ALA but can make EPH and DHA from ALA
trans fatty acids
unnatural trans configuration of double bonds
formed by partial hydrogenation of unsaturated fatty acids to increase shelf-life (margarine) or stability at high temperature (deep-frying oils)
found in dairy products and meats
associated with high heart disease: increase LDL and lower HDL
melting point is in-between unsaturated and saturated fatty acids because of its extended conformation
omega-3 fatty acid nomenclature
the carbon of the methyl group (most distant from the carboxyl group) is called the omega carbon and is given the number 1
the positions of the double bonds are indicated relative to the omega carbon
triacylglycerols
aka triglycerides or fats
most common and simplest lipid formed from 3 fatty acids in ester linkage with a glycerol
- simple triglycerides: same fatty acids
- mixed triglycerides: most naturally occurring; mixed fatty acid chains
insoluble and less dense than water (fats and oils float)
function: store energy; provide insulation
wax
esters of long-chain saturated and unsaturated fatty acids with long-chain alcohols
insoluble and higher melting points than triglycerides
function:
- store fuel in plankton
- protect hair and skin in vertebrates and keep it pliable
- waterproofing of feathers in birds
- protection from evaporation in tropical plants and ivy
- used by people in lotions, ointments, and polishes
glycerophospholipid
aka phosphoglycerides
primary constituents of cell membranes
2 fatty acids form ester linkages with glycerol and one highly polar/charged group is attached via a phosphodiester linkage to the third carbon
different organisms and tissues have different membrane lipid head group compositions; the head groups determine the surface proteins of membranes
lots of diversity possible by modifying backbone or the head groups, and changing fatty acids
- phosphatidic acid
- phosphatidylserine
- phosphatidylcholine: a major component of most eukaryotic cell membranes; many prokaryotes like E. Coli cannot synthesize this lipid
Ether Lipids
ether analogs of glycerophospholipids
plasmalogen: ether analog of phosphatidylethanolamine
- common in vertebrate heart tissue (heart is about 50% ether lipids)
- also found in some protozoa and anaerobic bacteria
- function not well understood: resistant to cleavage by common lipases (only cleaved by few specific); increase membrane rigidity; sources of signaling lipids; may be antioxidants
platelets-activating factor: ether analog of phosphatidylcholine
- acetic acid has esterified position C2
- signaling lipid
- plays an important role in inflammation and allergic response
- stimulates aggregation of blood platelets
sphingolipids
the backbone is not glycerol; it’s sphingosine
ceramide is the structural parent of all sphingolipids
3 subclasses (ceramide derivatives): sphingomyelins (phosphocholine), glycosphingolipids (sugars), and gangliosides (oligosaccharides + sialic acid)
sphingomyelins
phosphocholine head group
structurally similar to phosphatidylcholine (glycerophospholipid) but phosphatidylcholine has 2 FAs and sphingomyelin has one
found in plasma membrane and myelin sheath
biologically active lipids
present in smaller amounts than storage or structural lipids
eicosanoids, steroid hormones, bile salts, fat-soluble vitamins (ADEK), and polyketides
eicosanoids
carry messages to nearby cells (signaling lipid)
arachidonic acid derivative; arachidonic acid is a metabolite of the omega-6 fatty acid (linoleate)
NSAIDs block the formation of eicosanoids like prostaglandins (inflammation and fever) and thromboxanes (formation of blood clots)
steroid hormones
carry messages between tissues (regulate gene expression)
made from cholesterol in gonads and adrenal glands; have steroid nucleus but lack the alkyl chain (more polar)
carried through bloodstream usually attached to carrier proteins
lipid diffusion
lateral diffusion: very fast at room temperature; uncatalyzed
transverse diffusion: very slow when uncatalyzed; spontaneous flips are rare because the charged head group must cross the hydrophobic tail region
- flippase: catalyze transverse diffusion of PE and PS from outer to cytosolic leaflet against the concentration gradient (use ATP)
- floppase: moves phospholipids from cytosolic to outer leaflet using ATP
- scramblase: moves lipids in either direction toward equilibrium; thermodynamically neutral process
oxidation-reduction reactions
many biochemical reactions involve the transfer (and acceptance) of two electrons and two protons in the form of a proton and a hydride ion; these reactions are commonly called dehydrogenations
oxidation reactions generally release energy; in many dehydrogenases, the reaction proceeds by a stepwise transfer electrons to carriers like NAD, which is reduced to NADH
pyridine nucleotides
NAD and NADP (common redox cofactors)
- only carriers that are soluble
- can dissociate from the enzyme after the reaction
- in a typical biological oxidation reaction, the hydride from alcohol is transferred to NAD+/NADP+, giving NADH/NADPH
- the second proton is released to the aqueous solvent
- formation of NADH can be monitored by UV spectrophotometry (measure change of absorbance at 340 nm)
flavin nucleotides
FMN and FAD: common redox cofactors (not soluble)
tightly bound to flavoprotein, which holds onto electrons while it catalyzes the electron transfer from a reduced substrate to an electron acceptor
fused ring structure undergoes reversible reduction, accepting either one or two electrons in the form of one or two hydrogen atoms (each atom an electron plus a proton); fully reduced forms (accept 2 electrons) are FADH2 and FMNH2
permits the use of molecular oxygen as an ultimate electron acceptor (flavin-dependent oxidases)
List glycolytic entry points for other carbohydrates
dietary glycogen/starch:
- amylase hydrolyze glycosidic linkages, producing short polysaccharide fragments
- eventually, make d-glucose
disaccharides
- sucrose: broken down to glucose and fructose by sucrase
- lactose: broken down to glucose and galactose by lactase
- hexoses and fructoses are phosphorylated by the appropriate kinase
Glycolysis: The Preparatory Phase
2 ATPs are invested to raise the free-energy content of the intermediates; all the metabolized hexoses are converted to 2 glyceraldehyde 3-phosphates
step 3 (with phosphofructokinase-1) is the committing step to glycolysis; fructose 1,6-bisphosphate is not used in other biochemical reactions and is committed to becoming pyruvate and yield energy
Glycolysis: The Payoff Phase
step 6: first energy-yielding step (NADH); use of inorganic phosphate allows for net production of ATP
step 7: substrate-level phosphorylation (oxidation of bisphosphoglycerate) by phosphoglycerate kinase yields 2 ATP
step 10: 2nd production of ATP via substrate-level phosphorylation; pyruvate tautomerization drives ATP production by lowering reaction product
fates of pyruvate
catabolic
- aerobic metabolism: oxidized to yield acetyl CoA, which is completely oxidized to CO2 by the citric acid cycle
- lactic acid fermentation: reduced to lactate; accept electrons from NADH to regenerate NAD+; necessary for glycolysis to continue (generate ATP)
- ethanol fermentation: two-step irreversible reduction to ethanol; pyruvate decarboxylase (humans don’t have) and alcohol dehydrogenase
anabolic: synthesis of alanine and fatty acids
Pentose Phosphate Pathway
glucose 6-phosphate is shuttled to this pathway instead of being converted to pyruvate in glycolysis
main products:
- NADPH
- electron donors during biosynthesis of fatty acids and steroids
- repair oxidative damage
- ribose 5-phosphate
- a biosynthetic precursor of nucleotides (DNA, RNA, and coenzymes)
energy scorecards for glycolysis and for gluconeogenesis?
gluconeogenesis very expensive; uses 4 extra high-energy nucleotides to drive the synthesis of glucose
glycolysis location, feedstock, products, and regulated enzymes?
location: cytoplasm of muscle and brain
main feedstock/substrate: glucose and 2 ATPs
final products (per glucose): 2 pyruvates; 4 ATPs; 2 NADH
regulated enzymes: hexokinase, phosphofructokinase, pyruvate kinase
gluconeogenesis
location, main feedstock, final products, and regulated enzymes
location: mitochondria, cytoplasm, ER (liver), sometimes in kidneys
main feedstock: pyruvate (or oxaloacetate, lactate, and glycerol)
final products: glucose 6-phosphate and glucose (in liver)
regulated enzymes: pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxykinase, fructose 1,6-bisphosphatase-1
What are the steps in gluconeogenesis that bypass the irreversible steps in glycolysis?
pyruvate carboxylase and PEP carboxykinase
- bypass pyruvate kinase: phosphoenolpyruvate to pyruvate
- convert pyruvate to oxaloacetate (in mitochondria) and then phosphoenolpyruvate (in mitochondria or cytosol)
- requires ATP and GTP
fructose 1,6-bisphosphatase-1
- bypass phosphofructokinase-1
glucose 6-phosphatase
- bypass hexokinase
››Pyruvate dehydrogenase complex
reaction, location, final products, cofactors, regulation
oxidative decarboxylation of pyruvate catalyzed by a huge (144 peptides) complex
location: mitochondrial matrix; requires transport of pyruvate from the cytosol into mitochondria
final products: acetyl-CoA, NADH, CO2
cofactors: TPP, lipoic acid, FAD, NAD+
regulation: allosteric and covalent modification
