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.

review of chemical kinetics
rate equations
first order S -> P
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
Transtion State Theory, or diagram of a molecular collision
simple case: identical bonds, same energy before and after collision (H collides with H2)
usual case: different bonds, different energy before and after collision
The slowest step (highest transition state energy) determines the overall rate of a multistep reaction.
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)
enzyme kinetics (Voet Ch 14)
E= enzyme, S = substrate, ES = complex, P=product, I = inhibitor
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
catalytic mechanisms (Voet Ch 15);
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)
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)
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)
F1,6bP + H2O -> F6P + Pi (fructose-1,6-bisphosphatase)
spending ATP to gain more control over the reaction
Every pathway has a first committed step (large negative delta G) so that you don’t waste energy making unneeded intermediates.
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.
Day 7
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 don’t 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.
overview and common metabolites
scary version
road maps version (Mathews book)
Stryer Conceptual Insights (click Ch22: Overview of Carb & FA metabolism)
thermodynamics of phosphate compounds
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.
Standard free energies of hydrolysis. Note that ATP is in the middle, which makes it a useful currency (ie, you can’t 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
individual pathways
why glucose? (Stryer Ch16.01)
glucose forms easily from formaldehyde (common in the prebiotic seas)
glucose is stable in ring form and thus does not attack protein amino groups
glycolysis (Voet Ch 17)
2 ATP in, 4 ATP + 2 NADH out
less efficient, but the enzymes are in high concentration, so it’s fast
step 3, phosphofructokinase, is the major control point
ATP inhibits (also citrate)
AMP enhances
going back to glucose requires bypassing PFK; Stryer Conceptual Insights (click Ch16: Energetics of Glucose Metabolism)
Krebs or Citric Acid Cycle (Voet Ch 20)
1 Acetyl-CoA in, 3 NADH, 1 FADH2, 1 GTP
it’s 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 OA
succinyl-CoA competes with Acetyl-CoA
step 3 isocitrate dehydrogenase
ADP or Ca++ increases isocitrate binding
ATP decreases it (noncompetitive)
step 4, alpha-ketoglutarate dehydrogenase
succinyl-CoA decreases
Ca++ increases
more on redox reactions (Voet Ch 22)
Oxidation< > reduction reactions transfer electrons (H+ often follows along tobalance the charges)
NAD+ + 2e- + 2H+ < > NADH (Stryer Ch14.3)
FAD + 2e- + 2H+ < > FADH2 (Stryer Ch25.5)
Oxidative phosphorylation is the real powerhouse of the cell (in the presence of oxygen)
1 NADH -> 3 ATP (30 ATP/ glucose oxidized)
1 FADH2 -> 2 ATP (4/ glucose oxidized)
vs. 2 net ATP from glycolysis alone (no oxygen required)
In eukaryotes, this takes place on the inner mitochondrial membrane. In prokaryotes, on the plasma membrane.
Final step, cytochrome c oxidase, is the control point; other steps operate near equilibrium (delta G ~ 0)
controlled purely by substrate availability (unusual)
high [NADH]/[NAD+] means high activity
low [ATP]/[ADP][Pi] means high activity
proteins (Voet Ch 26)
Nitrogen is removed and disposed of
Carbon skeletons enter citric acid cycle (Voet Ch 20)
nucleic acids (StryerCh20.3)
NADH is used in energy generation [NAD+]/[NADH] ~ 1000
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
lipids (Voet Ch 25)
break down in 2-carbon units to form Acetyl-CoA, which enters citric acid cycle
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
Day 8
read MIT chapter Prokaryotic Genetics and Gene Expression
read Stryer Chapter 31: Control of Gene Expression

Learning Objectives: The major points from today are that 1) bacteria use specific protein repressors to hold RNA polymerase away from currently unneeded sets of genes. 2) Eukaryotes use a general repression mechanism (coiling of DNA onto histone proteins) and specific transcription factor proteins to recruit RNA polymerase onto the DNA. 3) Genes can regulate their own transcription and the transcription of other genes, based on internal dynamics (as in the circadian oscillator below) or based on signals from outside the nucleus, which we will see tomorrow.
Expression Control in Prokaryotes
promoter: a DNA sequence that binds tightly to RNA polymerase
a protein that binds to DNA, reducing or preventing RNA polymerase binding
review: making RNA (RNA polymerase) (Stryer Ch28.1)
initiation (Stryer Fig5.4)
promoters with more G-C content are transcribed less often
Genetic Networks in Prokaryotes
lactose, tryptophan and arabinose operons (the operons U Az)
lac from Access Excellence (Mathews Ch26lor)
trp from Access Excellence (Mathews Ch26lara)
ara (Griffiths Ch14.5) (Mathews Ch26ao)
Expression Control in Eukaryotes (Stryer Ch31.2)
promoters & transcription factors; note that in prokaryotes, transcription happens if not repressed, whereas in eukaryotes, it must be activated.
a DNA sequence with no promoter activity of its own, but that increases the activity of an associated promoter by perturbing local chromatin structure
DNA methylation prevents transcription in SOME eukaryotes; for instance, Drosophila the fruit fly is not methylated at all.
Genetic Networks in Eukaryotes
Circadian Rhythm network by Paul Smolen at UT-Houston (Biophys J)
note the positive feedback loop
usually contained inside a negative feedback loop to maintain stability
CREB gene network in memory

Day 9
read MIT chapter Cell Biology, subsections 8-12; Stryer Chapter 15: Signal Transduction Pathways

Learning Objectives: 1) To understand how a multistep signaling cascade leads to both fine control over individual enzymes (as we saw in metabolic cascades) and to quick signal amplification. 2) To understand the difference between specific local signals such as protein phosphorylation and general, global variables (which are now frowned upon in computer programming) such as the concentration of calcium, ATP, or NADH.
Intracellular Signaling
getting across the membrane
steroid hormones diffuse across membranes (Stryer Ch31.3)
G-proteins diffuse within the membrane Stryer Conceptual Insights (click Ch32: Signal Pathways Response & Recovery)
ion channels are anchored in the membrane
voltage sensors
evolutionary relationships (Goldin article)
NMDA channel is both ligand- and voltage-gated; acts as a coincidence detector (Purves Fig25.9)
cytoplasmic amplification and integration
kinase/phosphatase cascades
glycogen metabolism (Voet Ch 18)
“pure” signaling examples (Voet Ch 19)
signaling kinases are a huge family unique to eukaryotes(Protein Kinase Evolution)
calcium (Purves Fig8.7)
stimulates muscle contraction
causes neurotransmitter release
increases ATP production (glycolysis and citric acid cycle)
contributes to cell death
redox signaling
Day 10
Today we finish out the second week with a discussion of how cells talk to one another. The details of the actual machinery are always different, and endlessly fascinating for those of us who like to collect facts. What we want to abstract out of those details here are the principles of interlocked feedback loops.
Learning Objectives: 1) To see that the differences in extracellular signaling are differences of mechanism and scale; hormones are stable, travel long distances, and have longer-term effects than second messengers inside cells. Neurons are optimized to span long distances very quickly. 2) To understand the principles of combinatorial coding, which efficiently allow a small number of ‘letters’ to produce a huge number of ‘words’, ‘sentences,’ or ‘books’.
read Lodish chapter 20: Cell-Cell Signaling
Combinatorial Control
Genetic Code: 4 bases ^ 3-base codons = 64 possibilities (20 used)
Proteins: 20aa ^ <1000-peptide chains = some unreasonably large number, of which only a small space are actually used, because domains are similar across many proteins
Signaling Cascades: 2 (on or off) ^ 3-4? phosphorylation sites per enzyme; also take into account multiple steps per pathway
Odor Receptors: # genes ^ # receptors each odorant molecule can stimulate
Cortical Neurons: 10^9 neurons ^ 10^4-5 synaptic connections/ neuron
Extracellular Signaling
hormones, diffusible blood-borne messengers (Endocrinology on Pubmed Bookshelf)
3 types, depending on how far they travel
autocrine, same cell that released it
paracrine, nearby cells
endocrine, faraway cells
insulin (51aa) decreases blood glucose
glucagon (29aa) increases blood glucose
3 chemical types
polypeptides (insulin)
steroids (estrogen, testosterone)
amino acid derivatives (epinephrine, norepinephrine, thyroxin)
hormones oppose one another and are arranged in the same types of loops as other signaling molecules (Voet Ch 18) (pituitary animation)
transmitters are stored in vesicles
amino acid derivatives (many are also hormones)
dopamine, norepinephrine, epinephrine (tyrosine derivatives)
serotonin, melatonin (tryptophan derivatives)
GABA (derivative of glutamate)
peptide transmitters
nitric oxide is an exception that can’t be stored in vesicles because it diffuses across membranes
release of vesicles is driven by calcium
pheromones affect other individual organisms