Week 2: Networks and Feedback Loops
The second big idea in biology is that life is formed of networks of interacting units that regulate one another. Information flows in both directions. This is true at every level of biological organization, from groups of genes at the molecular level to groups of organisms at the ecological level. We will see feedback loops, both positive and negative, in examples from networks of genes, metabolic enzymes, and signaling molecules.
Day 6
Read MIT chapter Enzyme Biochemistry
Learning Objectives: 1) To understand why the rates of reactions depend on energy barriers or transition states. 2) To understand the basic mechanism of catalysis and recognize the signatures of different types of enzyme inhibition. 3) To identify control points in biochemical cascades by both kinetic and thermodynamic measures.
Catalysis
review of chemical kinetics
most enzymes use more than one mechanism; also note the possibility of channeling substrate through the body of the enzyme from one active site to another, so that it cannot diffuse away (Stryer Fig25.5)
rate equations
enzyme kinetics (Voet Ch 14)
first order S -> P
Transtion State Theory, or diagram of a molecular collision
second order S1 + S2 -> P
third order S1 + S2 + S3 -> P are rare because they require the simultaneous collition of three molecules; THIS IS ONE REASON BIOCHEMICAL REACTIONS REQUIRE SO MANY STEPS
fourth order essentially never happen
simple case: identical bonds, same energy before and after collision (H collides with H2)
The slowest step (highest transition state energy) determines the overall rate of a multistep reaction.
usual case: different bonds, different energy before and after collision
Catalysts reduce transition state energy
10-fold enhancement from 5.71kJ/mol (one H-bond)
million-fold enhancement from 34.25kJ/mol (less than one covalent bond)
E= enzyme, S = substrate, ES = complex, P=product, I = inhibitor
catalytic mechanisms (Voet Ch 15);
catalysis E + S < > P + E
competitive inhibition: inhibitor binds to enzime active site, displacing substrate
E+I < > EI -> no reaction
noncompetitive inhibition: inhibitor binds directly to ES complex but not to the free enzyme: remember that binding and catalysis can take place at different sites on the same enzyme
E+S < > ES + I < > ESI -> no reaction
mixed inhibition: both the free enzyme and the ES complex bind the inhibitor
acid-base catalysis
: adding/removing H+ stabilizes the transition state
acidic or basic amino acids
covalent: transient formation of a catalyst-sustrate covalent bond
Lys, His, cys, Asp, Ser
metal ion: like protons, but stable at neutral pH and charges >+1
electrostatic: excluding water from the active site changes the local dielectric constant, because water shields charges; this changes the pK of active site amino acids
proximity and orientation effects: stop or slow relative motions (bond rotations, etc.), decreasing local entropy, and provide proper orientation
preferential binding of the transition state: stabilizing the transition state relative to the substrate;
Flux Measurements
free energy of cascades Stryer Conceptual Insights (Click Ch14: Energetic Coupling)
Day 7
control of metabolic flux (J = forward - reverse)
Pathways are irreversible (large negative delta G), but a separate enzyme pathway can get around this. Stryer Conceptual Insights (Click Energetics of Glucose Metabolism)
F6P + ATP -> F1,6bP + ATP (phosphofructokinase)
Every pathway has a first committed step (large negative delta G) so that you dont waste energy making unneeded intermediates.
F1,6bP + H2O -> F6P + Pi (fructose-1,6-bisphosphatase)
spending ATP to gain more control over the reaction
The reason these enzymes operate so far from equilibrium (have a large negative delta G) is that they are slow. They do not have time to equilibrate substrates with products because a faster enzyme further down the path drains products away. THESE SLOW ENZYMES ARE THE CONTROL POINTS.
read Stryer chapter 14: Metabolism, MIT chapter Glycolysis and the Krebs Cycle
Last week we started our study of large molecules (polymers) by looking at their subunits (monomers) and the bonds that held them together. Then we saw that these individual monomers interacted through hydrogen bonds or hydrophobic forces to produce other, new, emergent properties such as secondary and then tertiary structure. This week we take the same approach. Yesterday we looked at individual enzymes. Today we look at multi-enzyme pathways to see how their interactions create higher levels of structure, in this case feedback loops. We dont have time to examine every reaction in detail (metabolism is usually at least two weeks in a biochem course), so we will focus on the large-scale relationships between metabolic pathways.
Learning Objectives: 1) To understand the relationhips between metabolic cascades by identifying their common intermediates and their common activators or inhibitors. These common points allow cascades to be controlled independently when necessary, or in concert with one another. 2) To understand why glucose and ATP in particular should be the energy currency of cells.
Metabolism
overview and common metabolites
thermodynamics of phosphate compounds
scary version
road maps version (Mathews book)
Stryer Conceptual Insights (click Ch22: Overview of Carb & FA metabolism)
An average woman burns 1500-1800kcal (6300-7500kJ) /day. That works out to over 200 mol ATP-> ADP + Pi, yet total [ATP] < 0.1mol. ATP is constantly recycled.
individual pathways
Standard free energies of hydrolysis. Note that ATP is in the middle, which makes it a useful currency (ie, you cant make change with a $100 bill). If ATP were at the top, how would you regenerate ATP?
phosphoenolpyruvate -61.9kJ/mol
phosphocreatine -43.1
PPi -33.5
ATP -> AMP + PPi -32.2
ATP -> ADP + Pi -30.5
glucose-1-phosphate -20.9
fructose-6-phosphate -13.8
glucose-6-phosphate -13.8
glycerol-3-phosphate -9.2
carbohydratesIn eukaryotes, this takes place on the inner mitochondrial membrane. In prokaryotes, on the plasma membrane.
why glucose? (Stryer Ch16.01)more on redox reactions (Voet Ch 22)
glucose forms easily from formaldehyde (common in the prebiotic seas)glycolysis (Voet Ch 17)
glucose is stable in ring form and thus does not attack protein amino groups
2 ATP in, 4 ATP + 2 NADH outKrebs or Citric Acid Cycle (Voet Ch 20)
less efficient, but the enzymes are in high concentration, so its fast
step 3, phosphofructokinase, is the major control point
ATP inhibits (also citrate)going back to glucose requires bypassing PFK; Stryer Conceptual Insights (click Ch16: Energetics of Glucose Metabolism)
AMP enhances
1 Acetyl-CoA in, 3 NADH, 1 FADH2, 1 GTP
its a closed loop, so intermediates are regenerated and only need to be present in tiny amounts like a catalyst
3 control points
step 1, citrate synthase
citrate competes for binding with OAstep 3 isocitrate dehydrogenase
succinyl-CoA competes with Acetyl-CoA
ADP or Ca++ increases isocitrate bindingstep 4, alpha-ketoglutarate dehydrogenase
ATP decreases it (noncompetitive)
succinyl-CoA decreases
Ca++ increases
Oxidation< > reduction reactions transfer electrons (H+ often follows along tobalance the charges)Oxidative phosphorylation is the real powerhouse of the cell (in the presence of oxygen)
NAD+ + 2e- + 2H+ < > NADH (Stryer Ch14.3)
FAD + 2e- + 2H+ < > FADH2 (Stryer Ch25.5)
1 NADH -> 3 ATP (30 ATP/ glucose oxidized)vs. 2 net ATP from glycolysis alone (no oxygen required)
1 FADH2 -> 2 ATP (4/ glucose oxidized)
controlled purely by substrate availability (unusual)proteins (Voet Ch 26)
high [NADH]/[NAD+] means high activity
low [ATP]/[ADP][Pi] means high activity
Nitrogen is removed and disposed ofnucleic acids (StryerCh20.3)
Carbon skeletons enter citric acid cycle (Voet Ch 20)
NADH is used in energy generation [NAD+]/[NADH] ~ 1000lipids (Voet Ch 25)
NADPH is used in synthesis [NADP+]/[NADPH] ~ 0.01
Allows for independent regulation of metabolism and biosynthesis
synthesis is inhibited by the products (Stryer Fig25.16); note that AMP inhibits nucleotide synthesis but increases glycolysis
break down in 2-carbon units to form Acetyl-CoA, which enters citric acid cycleDay 8
major control point is Acetyl-CoA carboxylase (Stryer ch25.5)
citrate speeds it up
palmitate slows it down
covalent phosphorylation turns it off (kinase is activated by AMP)
product, Malonyl-CoA, inhibits transport of fatty acyl-CoAs into the mitochondrion, therefore slowing oxidation
promoter:a DNA sequence that binds tightly to RNA polymerase
initiation (Stryer Fig5.4)
promoters with more G-C content are transcribed less oftenGenetic Networks in Prokaryotes
lactose, tryptophan and arabinose operons (the operons U Az)Expression Control in Eukaryotes (Stryer Ch31.2)
lac from Access Excellence(Mathews Ch26lor)
trp from Access Excellence (Mathews Ch26lara)
ara (Griffiths Ch14.5) (Mathews Ch26ao)
promoters & transcription factors;note that in prokaryotes, transcription happens if not repressed, whereas in eukaryotes, it must be activated.
Circadian Rhythm network by Paul Smolen at UT-Houston (Biophys J)
note the positive feedback loopCREB gene network in memory
usually contained inside a negative feedback loop to maintain stability
getting across the membrane
steroid hormones diffuse across membranes (Stryer Ch31.3)cytoplasmic amplification and integration
G-proteins diffuse within the membrane Stryer Conceptual Insights (click Ch32: Signal Pathways Response & Recovery)
ion channels are anchored in the membrane
ligand-gated
voltage-gated
voltage sensorsNMDA channel is both ligand- and voltage-gated; acts as a coincidence detector (Purves Fig25.9)
evolutionary relationships (Goldin article)
kinase/phosphatase cascadesDay 10
glycogen metabolism (Voet Ch 18)calcium (Purves Fig8.7)
pure signaling examples (Voet Ch 19)
signaling kinases are a huge family unique to eukaryotes(Protein Kinase Evolution)
stimulates muscle contractionredox signaling
causes neurotransmitter release
increases ATP production (glycolysis and citric acid cycle)
contributes to cell death
Genetic Code: 4 bases ^ 3-base codons = 64 possibilities (20 used)
hormones, diffusible blood-borne messengers (Endocrinology on Pubmed Bookshelf)
3 types, depending on how far they travel
autocrine, same cell that released it3 chemical types
paracrine, nearby cells
endocrine, faraway cells
insulin (51aa) decreases blood glucose
glucagon (29aa) increases blood glucose
polypeptides (insulin)hormones oppose one another and are arranged in the same types of loops as other signaling molecules (Voet Ch 18) (pituitary animation)
steroids (estrogen, testosterone)
amino acid derivatives (epinephrine, norepinephrine, thyroxin)
transmitters are stored in vesicles
amino acid derivatives (many are also hormones)release of vesicles is driven by calcium
dopamine, norepinephrine, epinephrine (tyrosine derivatives)peptide transmitters
serotonin, melatonin (tryptophan derivatives)
glycine
glutamate
GABA (derivative of glutamate)
acetylcholine
nitric oxide is an exception that cant be stored in vesicles because it diffuses across membranes
pheromones affect other individual organisms