Unit 2 Biology

Metabolism

A set of linked reactions that degrade fuel molecules and construct biomolecules

What do cells use ATP for?

1) Synthesis of macromolecules

2) Signal transduction and active transport of molecules of ion

3) Mechanical work or muscle movement

Phototrophs + Examples

Obtain energy from sunlight. Use photosynthesis. Examples include plants and cyanobacteria.

Chemotrophs + Examples

Obtain energy from oxidation of carbon fuels. Fuel sources come from sugars (glucose and glycogen,) lipids (fatty acids stored at triglycerides,) and proteins (during starvation). Examples include plants, animals, and bacteria.

Catabolic Pathway

Breakdown of carbon “fuels” to synthesize ATP. Exergonic pathways with energy as a product. EX: Glycolysis

Anabolic Pathway

Uses ATP to synthesize larger biomolecules. Endergonic pathways that require energy. Energy is captured in chemical bonds. EX: Photosynthesis, protein synthesis

Catabolism and Anabolism

The energy harvested as ATP during the break down of molecules in catabolic pathways can be used to synthesize molecules in anabolic pathways

ATP Structure

Base (adenine), ribose (5-carbon sugar), 3 phosphates (2 phosphoanhydride bonds), and one gamma phosphate is one of the end.

Phosphoanhydride bonds

High energy bonds in ATP that store chemical energy (potential energy).

Energetic Coupling of Reactions

The exergonic nature of ATP hydrolysis coupled to an endergonic reaction can result in an overall exergonic reaction. EX: Transfer of the gamma phosphate to a substrate

Step 1 of Glycolysis

Exergonic reaction of ATP hydrolysis can be used to create a high energy molecule used in glycolysis

Energy Charge of the Cell Used to Regulate Pathways

Catabolic pathways activated when the energy charge is low in the cell. Anabolic pathways activated when energy charge is high in the cell.

Energy Charge

Reflects the energy status of the cell

High Energy Molecules

Include PEP, (1,3- BPG), and creatinine phosphate

Creatine Phosphate

Creatine phosphate is used by muscle cells. It is even more exergonic than ATP and can be used to drive generate ATP is muscle cells. Creatine Phosphate + ADP —> ATP +Creatine

Glucose Oxidation Reaction

C6H12O6 + 6O2 —> 6H2O + 6CO2 + energy. Similar to a combustion reaction. Used to generate ATP

Oxidation and Reduction

Oxidation and reduction go together. Oxidation is the loss of electrons, reduction is the gain of electrons.

Oxidation

Carbon has more bonds to oxygen. Often associated with losing a hydride (H+ + 2e-) or with gaining a bond to oxygen

Reduction

Carbon has more bonds to hydrogen. Often associated with gaining a hydride (H+ + 2e-) or with gaining a bond to hydrogen

Glycolysis Overview

10 enzymatic steps converting 1 molecule of glucose to 2 pyruvate. First 5 steps are energy investing, second 5 steps are energy harvesting

Where does glycolysis occur?

Cytoplasm

What does glycolysis produce?

2 ATP, 2 pyruvate, 2 NADH, 2 water

First 5 Steps of Glycolysis

5 steps, net 2 ATP. Creates 2 molecules of GAPs. Step 3 commits to glycolysis via Phosphofructokinase (PFK).

Step 1 Glycolysis

Hexokinase catalyzes the reaction between ATP and glucose to make glucose-6-phosphate

Hexokinase

• Hexokinase closes once both substrates are bound

• Closes out water molecules from the aqueous cytoplasm

• Brings substrates in close proximity to react

• Mg²+ in the active site stabilizes ATP

Steps 5-10 (Energy Harvesting Reactions)

ATP is produced at 2 different steps (7 and 10). NADH is produced (step 6). Pyruvate (2) are produced

Carbon oxidation

Coupled with reduction of an electron carrier named NAD+

Regulation of Irreversible Steps

Phosphorylation, feedback inhibition and/or allosteric inhibitors, transcription regulation, enzyme degredation

Reversible Steps

Typically not regulated and determined by substrate v product concentrations

Phosphofructokinase

• Irreversible reaction

• Has two substrates: ATP and fructose 6 phosphate

• ATP is both a substrate and allosteric regulator

  • Under low ATP concentrations, ATP only binds to the active site

  • Under high ATP concentrations, ATP binds to the allosteric site and prevents phosphorylation

Km in PFK

Low in active site, high in allosteric site

Fermentation Pathway

• Active when no oxygen is present

• Cellular purpose is to regenerate NAD+ so cells can continue to glycolysis

• Mammalian fermentation pathways produce lactic acid

• Fermentation pathways in yeast and bacteria generate useful products such as acetic acid, ethanol, acetone, yogurt and cheese`

Fate of Pyruvate

Aerobic Respiration or anaerobic respiration

Oxidation of Pyruvate

• Occurs in the mitochondrial matrix

• Creates 2 Acetyl CoA, 2 CO2, and 2 NADH

• Catalyzed by pyruvate dehydrogenase

Acetyl-CoA

Acetyl-CoA is a central metabolite that is in common with fatty acid degradation, amino acid degradation, and sugar metabolism

Mitochondria Structure

• Outer membrane contains pores permeable to small molecules

• Inner membrane is highly folded to form cristae, where ETC takes place

• Matrix is the site of pyruvate oxidation and citric acid cycle

• Intermembrane space low pH, high H+

Last Step of CAC

Malate is oxidized to oxaloacetate

What are the products of the CAC?

4 CO2, 6 NADH, 2 FADH, 2 ATP

Diagram Glycolysis, Pyruvate Oxidation, CAC. What does each step generate and where does each step happen?

ETC

• Flow of electrons from high to low free energy

• Complex I: NADH donates electrons and pumps H+ into intermembrane space

• Complex II: FADH donates electrons but none are pumped into the intermembrane space

• Complex III: H+ are pumped into the intermembrane space

• Complex IV: H+ is pumped into the intermembrane space, O2 is reduced to H2O

• Complex V (ATP Synthase): Protons flow down gradient and synthesize ATP

NADH vs FADH2

Both carry 2 electrons. However, NADH generates 2.5 ATP/e- pair while FADH2 generates 1.5 ATP/e- pair because NADH donates to the ETC at a higher energy level

Chemiosmotic Hypothesis

Proton gradient (from the intermembrane space to the matrix) provides the energy needed to make ATP

Adipose Tissue

Stores and releases fatty acids

Liver

Maintains constant level of blood glucose

Brain

Requires glucose and oxygen (ketone bondies during starvation)

Muscle

Stores glycogen, metabolizes FA

Insulin

Stimulates organs that can store glucose to take it out of the blood and store it as either glycogen or lipids

Glucagon and epinephrine

Reverse the effects of insulin and act to mobilize glucose and increase glucose in the blood

ATP Synthase Structure

• Made of 8 subunit types

• Subunits group to form two main component F0 and F1

F0

Proton Channel

1) Rotation of the c ring rotates the gamma subunit

2) The irregular shape of the gamma subunit forces conformational changes of the beta subunits: Open (O), Loose (L), and Tight (T0)

3) Chemical energy of proton gradient is converted to mechanical energy of rotating c ring, which is converted to chemical energy in ATP

F1

An enzyme wheel in the mitochondrial matrix that catalyzes ATP synthesis from ADP and P

Aspartic Acid

Proton enters subunit a from the intermembrane space and moves to a subunit c helix. Subunit c contains aspartic acid which holds the H+ from the proton gradient, becomes neutralized and can rotate

Beta Subunits

Result in ATP Synthesis

Open: ADP, Pi, and ATP free to diffuse in and out of B subunit

Loose: ADP+Pi trapped

Tight: ADP+Pi converted to ATP in yellow B subunit

ATP Synthase Stoichiometry

One 360 degree rotation of gamma results in 3 units

Photosynthesis Reactions

6CO2+12H2O+light energy —>C6H12O6+6H2O+6O2, involve light independent and light dependent reactions

Where does photosynthesis take place?

Mesophyll cells with chloroplasts

Where do the light dependent reaction occur?

Thylakoid membrane

Absorption Spectra for Chlorophyll

A and b

Photosystem

Absorbs a photon, excites an electron (oxidized) which gets accepted by an electron acceptor

Thylakoid lumen

Analogous to intermembrane space, high H+ concentration

Cytochrome

Passes electrons

H2O and O2 in photosynthesis

H2O is oxidized to O2 which diffuses across membrane

NADPH

Contains a phosphate group so that it won’t be used in glycolysis

Z Scheme

Photosystem II (P680) —> Cytochrome complex —> Photosystem I (P700) —> FD-NADP+ Reductase —> NADPH

Calvin Cycle

Location: Stroma

Inputs: NADPH, ATP, CO2

Outputs: GAP, O2, ADP, NADP+

3 Stages of Calvin Cycle

1) Carboxylation (add carbon to RuBP, degrades into 2 3 carbon molecules)

2) Reduction

3) Regeneration of RuBP

Carboxylation

CO2 is combined with RuBP via rubisco, unstable 6 carbon intermediate that splits into 2 × 3 carbon molecules

ETC Disruption

Atrazine blocks e- transfer at PS II

Paraquat blocks e- transport at PS1

Stoichiometry of Calvin Cycle

6 Carbon Enter, there are 6 RuBP molecules so 30C toatl. Go to 36C total (12 3PG) —> 12 G3P. Two G3P leave the cell as sugars (one glucose -6C). Rubisco generation

C3 Plants

CO2 is fixed directly by the enzyme rubisco in mesophyll cells. When O2 levels are high in hot conditions, photorespiration occurs.

C4 Plants

CO2 first enters in mesophyll cells. The enzyme PEP carboxylase fixes CO2 into a 4-carbon molecule. The 4 carbon molecule moves to the bundle sheath cells.

Central Dogma

DNA —> RNA —> Protein

Griffiths Experiment

S strain killed the mouse

Heat killed S strain did not kill the mouse

Heat killed mixed with R strain killed the mouse, must have been a transformation

Griffiths Experiment Techniques

1) Separated components of the heat killed S strain to see if transformation was present

2) Used chemicals/enzymes to destroy fractions that had the transforming property to see if that property could be specifically destroyed

Nucleotide Structure

Contains a sugar, nitrogenous base, and phosphate

5 carbon sugars (Draw)

Differ on the 2’ carbon, ribose contains a hydroxyl group, DNA does not

Purine and Pyramidens

Purine: Contains 2 rings. Includes Adenine and Guanine

Pyrimidines: Contain 1 ring. Include Thymine, Cytosine, and Uracil

Thymine and Uracil

Thymine contains a methyl group

Structure of Nucleotides

The base is always attached to the C1’ of the sugar via a B-glycosidic linkage

Sugar-Phosphate Backbone Structure

Polarity goes from 3’ —> 5’. Anti-parallel because of hydrophobic interactions’. Helix Nucleotide backbone connected via 3’-5; phosphodiester bonds

B-Form Helix

Contains 10.5 bp per turn

Right handed helix

Has major and minor grooves where DNA binds to

Linus Pauling

Proposed that DNA had a helix of 3 strands, bases on the outside and phosphate groups on the inside

DNA synthesis in eukaryotes vs prokaryotes

In prokaryotes: Happens continuously

In eukaryotes: There are checkpoints

Methods of DNA Replication

1) Semi Conservative

2) Conservative

3) Dispersive

Explain the Experiment and Results for Each

DNA Replication

1) DNA polymerase catalyzes the polymerization of deoxyribonucleotide triphosphates (dNTPs)
2) Requires a single-stranded DNA template

3) Newly synthesized strand must be antiparallel to the template strant. 5’ —> 3’ direction

4_ Requires a primer to a 3’ -OH group

Helicase

Separates DNA strands, allowing DNA polymerases access to single-stranded templates. Requires ATP hydrolyses

Single-stranded binding protein (SSBP)

Binds ssDNA after helicase, holds strands apart

Primase

Synthesizes a short (~10 nucleotides) RNA primer

Provides a 3’ -OH group for DNA polymerases to begin synthesis

Okazaki Fragments

Occur due to the 5’ to 3’ polymerizatiom

DNA Polymerase I

Digests the RNA primer (5’ —> 3’) and fills in the gap

DNA Ligase

Nick sealed by DNA ligase

Initiation of DNA Transcription

Starts at oriC. DnaA hexamer binds to binding sites. Lots of AT base pairs

Draw Replication (two sided with helicase)

Plasmid

Small circular extrachromosomal DNA molecules that replicate independently of the bacterial host chromosome and have fixed origions of replications (ori)