Exam 2- Chapter 14- Cellular metabolism

Question 1

ATP synthase

Answer 1

Also called F0F1 ATPase. It is an F-type membrane pump with 2 domains (called F0 and F1, hence the alternate name). ATP synthase opens a pathway for protons (H+) to travel down their gradient into the cytosol (the mitochondrial matrix). The energy released from protons moving down their gradient is used to drive an energetically unfavorable reaction between ADP and an inorganic phosphate- ATP synthesis. ATP synthase harnesses the energy and undergoes conformational changes to physically make ATP

Question 2

ATP synthase structure

Answer 2

A multisubunit protein that is greater than 500,000 Daltons. It has an upside down lollipop shape. The enzymatic portion (the lollipop head) is a ring of 6 subunits that projects into the cytosol, and the “stick” is stuck in the membrane. The enzymatic portion is the part that is actually creating ATP.

Question 3

Stator

Answer 3

The stationary portion of the ATP synthase. It is an elongated arm that tethers the head to a group of transmembrane proteins. It creates a channel through which the protons will travel- contains entry and exit channels for protons

Question 4

Rotor

Answer 4

The stator contacts the rotor- a ring of 10-14 identical transmembrane subunits. This is the part of ATP synthase that moves. Protons bind to the rotor as they travel down their concentration gradient, which moves the rotor around in a circle. The rotor moves up into the enzymatic portion, which will undergo conformational changes

Question 5

ATP synthase function

Answer 5

Works by rotary catalysis. Protons travel down their gradient through a channel, which is formed by contact between the stator and the rotor. Proton movement provides energy, causing the rotor ring to spin. Rotor spinning turns stalk/shaft which turns rapidly inside the lollipop head (enzymatic portion) creating repeated changes in protein conformation. 3 of its 6 subunits have binding sites for ADP and inorganic phosphate, which is important for creating ATP. This mechanical energy is converted into chemical bond energy to form the bond between ATP and inorganic phosphate

Question 6

F0 domain of ATP synthase

Answer 6

Embedded in the inner mitochondrial membrane

Question 7

F1 domain of ATP synthase

Answer 7

Enzymatic portion in the mitochondrial matrix

Question 8

ATP synthase mechanism (6 steps)

Answer 8

1. Protons pass through the channel formed by rotor (c) :stator (a) contact, moving down their concentration gradient.
2. As the protons travel, they bind to the rotor (10-14 C subunits), providing energy for the spinning motion of the rotor
3. H+ exit through channel in the stator after the rotor has undergone one full rotation
4. Rotor spinning turns the stalk (shaft) portion of the synthase
5. The spinning is transduced to the enzymatic portion. As the stalk (shaft) spins within the enzymatic portion, it is causing conformation changes in the 3 alpha and 3 beta subunits of the enzymatic portion, which drives ATP synthesis
6. ADP and inorganic phosphate are forced together to form ATP

Question 9

Beta subunits of ATP synthase

Answer 9

Contain nucleotide binding sites and catalytic domains. This is primarily where ADP and inorganic phosphate will bind, and they will be forced together to create ATP

Question 10

Side effects of energy production

Answer 10

Although metabolic processes are incredibly important to the cell and provide ATP, these processes also produce molecules that are toxic to the cell- this includes advanced glycation end products (AGEs) and reactive oxygen species (ROS)

Question 11

Advanced glycation end products (AGEs)

Answer 11

Irreversible addition of sugars to proteins or lipids- however, it is not the same thing as glycosylation. Glycosylation is the enzyme controlled addition of sugars to specific sites on proteins or lipids, which is usually necessary for their function. In contrast, glycation is a non-enzymatic, random addition of sugar to proteins or lipids. This is damaging and impairs function- these molecules were never supposed to have sugar as part of their structure

Question 12

Where are AGEs found?

Answer 12

They are induced by heating sugars with proteins or lipids. Therefore, they are found in fried foods and grilled meats- not dangerous in foods, just in cells

Question 13

How are AGEs considered side products of glycolysis?

Answer 13

Glycolysis forms glucose intermediates. At step 5 of glycolysis, where the path splits into 2 parallel pathways, glucose can undergo changes to its structure and become glyceraldehyde 3-phosphate or DHAP. Both compounds are susceptible to fragmentation (losing part of their structure), mainly through removal of a phosphate. When glucose loses part of its structure (a phosphate detaches), it becomes a highly reactive sugar called methylglyoxal. It can randomly attach itself to proteins in the cell, creating AGEs and impairing function as the protein structure changes. The protein becomes nonfunctional and toxic.

Question 14

Methylglyoxal lysine dimer (MOLD)

Answer 14

Methylglyoxal predominantly interacts with lysine amino acids, so typical damage is the formation of a lysine dimer (MOLD). Methylglyoxal is the added sugar and contributes to an imidazolium crosslink. This creates a dimer where one shouldn’t have existed before. This is an unregulated process that compromises the structure and function of proteins

Question 15

Glyoxalase system

Answer 15

An endogenous defense against the formation of AGEs. Glyoxalase I produces S-lactoylglutathione from methylglyoxal and GSH. Glyoxalase II produces D-lactate & GSH. This makes methylglyoxal into something harmless. The glyoxalase system is usually enough to stop too many AGEs from being produced. In situations of cell death, there can be hyperproduction of AGEs

Question 16

Reactive oxygen species (ROS)

Answer 16

Species of oxygen that react with other molecules (proteins, lipids) and damage them. Includes hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radical. Hydrogen peroxide is the least damaging of all the ROS because it can destabilize certain molecules. Free radicals are more dangerous

Question 17

Free radicals

Answer 17

Superoxide anions and hydroxyl radicals are the most dangerous ROS. They are referred to as free radicals because they have an unpaired electron. Because they have an unpaired electron, they need to steal another electron from another molecule, regardless of where it is. Stealing the electron makes the target molecule into a free radical, which therefore creates a chain reaction of damage and radical formation. More commonly, if a protein loses an electron, it will form a covalent bond to compensate. This is compromises the structure and function of the protein because the bond is not supposed to be there- the protein is nonfunctional at this point

Question 18

What happens to a protein when it is damaged by ROS?

Answer 18

Most commonly, if a protein loses an electron, it will form a covalent bond to compensate. The bonds can be disulfide or other types of covalent bonds. This is compromises the structure and function of the protein because the bond is not supposed to be there- the protein is nonfunctional at this point. There are multiple complications of unregulated covalent bond formation- the protein can lose function, unfold, be cleaved, or aggregate with other proteins

Question 19

Formation of ROS

Answer 19

Side product of the ETC in oxidative phosphorylation. Cytochrome C oxidase, which has the final electron acceptor of oxygen, can release oxygen too early, before it has taken all 4 electrons. As a result, a superoxide anion can be formed- this is the main way that ROS form. A superoxide anion may also be formed when electrons leak from NADH dehydrogenase and are picked up by oxygen (reverse electron transport)

Question 20

Reverse electron transport

Answer 20

When electrons move backward in the electron transport chain, back towards the first respiratory complex. This can result in ROS formation. A superoxide anion may be formed when electrons leak from NADH dehydrogenase and are picked up by oxygen

Question 21

Other aspects of the mitochondria (4)

Answer 21

1. They contain their own DNA
2. They can transcribe RNA and translate proteins- mRNA, rRNA, tRNA, and ribosomes. This is not well understood
3. Import most of their proteins from the cytoplasm
4. Import most of their lipids

Question 22

Mitochondrial protein import

Answer 22

Proteins imported into the mitochondria are usually taken up from the cytosol within seconds after release from the ribosomes. They are synthesized as mitochondrial precursor proteins in the cytosol. The proteins must be fully synthesized before they are taken up by the mitochondria, translocation always occurs post translationally. Mitochondrial translocation can be more complex than ER translocation due to the mitochondria having 2 membranes

Question 23

Complexes utilized for mitochondrial protein import (4)

Answer 23

1. Translocase for outer membrane (TOM) complex
2. Translocase for inner membrane (TIM) complex
3. Sorting and assembling machinery (SAM) complex
4. OXA complex

Question 24

Translocase for outer membrane (TOM) complex

Answer 24

Transfers proteins across the outer membrane of the mitochondria. It can move proteins into the intermembrane space and also assist with inserting proteins into the outer membrane

Question 25

Translocase for inner membrane (TIM) complex

Answer 25

Transfers proteins across the inner membrane of the mitochondria. There are 2 different TIMs- TIM23 and TIM22.

Question 26

TIM23

Answer 26

Moves proteins into the mitochondrial matrix, can also create transmembrane proteins.

Question 27

TIM22

Answer 27

Specialized for inserting specialized membrane proteins, including the ADP/ATP transporter

Question 28

Sorting and assembling machinery (SAM) complex

Answer 28

A specialized complex that handles beta barrel proteins imported by the TOM complex. It helps them to fold and insert properly in the outer membrane

Question 29

OXA complex

Answer 29

Insertion of proteins into the inner mitochondrial membrane when these proteins were made in the mitochondrial matrix

Question 30

Outer mitochondrial membrane protein importers (2)

Answer 30

1. TOM complex
2. SAM complex

Question 31

Inner mitochondrial membrane protein transporters (3)

Answer 31

1. TIM23 complex
2. TIM22 complex
3. OXA complex

Question 32

Mitochondrial protein translocation

Answer 32

Precursor proteins are translocated in an unfolded form. There are many dedicated chaperones for mitochondrial precursor proteins, like the Hsp70 family chaperones. The chaperones prevent proteins from misfolding and also help to push the proteins through the pore. Signal sequences bind to import receptors of the TOM complex in the mitochondrial outer membrane. Once translocation begins, chaperones are stripped off and unfolded. The polypeptide is fed into the channel, with the signal sequence entering first

Question 33

Hsp70 family proteins

Answer 33

Chaperone proteins that prevent proteins from misfolding and push the proteins through the pore. Goes through repeated rounds of ATP binding, hydrolysis, and ADP release to undergo repeated conformational changes and therefore push the protein across the membrane

Question 34

What happens after a protein is pushed through the TOM complex?

Answer 34

The protein is in the intermembrane space, and the membrane potential helps to attract the protein to the TIM complex- the intermembrane space is more positive. Mitochondrial Hsp70 will now help to pull the protein into the matrix

Question 35

Hsp60 proteins

Answer 35

Mitochondrial chaperone proteins found in the matrix. They assist the proteins with correct folding.

Question 36

Hsp60 mechanism (6)

Answer 36

1. Hsp60 is a barrel-like complex that captures unfolded proteins through hydrophobic interactions.
2. The protein is fed into the center of the barrel. A GroES “cap” is added on top of the barrel
3. ATP and cap binding expand the rim of the barrel- this is a conformational change that stretches/unfolds the client protein
4. Protein folding is catalyzed
5. ATP hydrolysis weakens the complex and new ATP binds, releasing the folded protein
6. If the protein is still incorrectly or not completely folded, more cycles will occur

Question 37

TOM complex transmembrane proteins

Answer 37

The TOM complex inserts alpha helical proteins into the outer membrane of the mitochondrial, similar to Sec61 in the ER (stop transfer and start transfer sequences). The TOM complex uses start and stop transfer sequences as well. With beta barrel proteins (porins), TOM can help to translocate them into the intermembrane space and the SAM complex will take over

Question 38

SAM complex transmembrane proteins

Answer 38

Main function is to translocate beta barrel proteins into the outer mitochondrial membrane. Once the TOM complex translocates the protein into the intermembrane space, chaperones will bind to the protein and prevent it from misfolding and escort it to the SAM complex. The SAM complex then arranges everything properly and inserts the protein into the outer mitochondrial membrane. The central SAM subunit is homologous to the bacterial outer membrane proteins that help to insert beta barrels from the periplasm- this is conserved through evolution

Question 39

TOM complex insertion of transmembrane proteins mechanism

Answer 39

A start transfer sequence indicates that the complex needs to start transferring the unfolded protein across the membrane. Once it encounters a stop transfer sequence, translocation stops. The protein moves laterally into the membrane, translocation begins again at a start transfer sequence and stops again at a stop transfer sequence. This creates a multipass alpha helical transmembrane protein in the outer mitochondrial membrane

Question 40

Single pass transmembrane protein in the inner mitochondrial membrane- mechanism

Answer 40

The translocation of the protein through the outer mitochondrial membrane continues until a stop transfer sequence is encountered. TOM pulls the remainder of the protein into the intermembrane space. The signal sequence is cleaved and the hydrophobic sequence it released from TIM23 into the membrane. TIM23 translocates the polypeptide directly into the matrix

Question 41

Direct translocation of inner membrane proteins into the matrix mechanism

Answer 41

TIM 23 can translocate polypeptides directly into the matrix- these are proteins destined for the inner membrane. The signal sequence is cleaved and the hydrophobic sequence is exposed. This hydrophobic sequences guides the protein to the OXA complex and the protein is inserted into the inner mitochondrial membrane

Question 42

Peripheral membrane proteins on the inner mitochondrial membrane mechanism

Answer 42

For peripheral membrane proteins, the hydrophobic sequence is cleaved after insertion. A TIM complex helps to insert the protein into the inner membrane, but the protein is peripherally associated with the membrane

Question 43

Multipass inner membrane proteins

Answer 43

TIM22 inserts these proteins similar to Sec61. This mechanism is similar to that of the TOM complex, start and stop transfer sequences are involved to move the protein through the membrane multiple times

Question 44

Endosymbiont hypothesis

Answer 44

The idea that mitochondria and chloroplasts probably evolved from bacteria. It is thought that some bacteria were endocytosed by eukaryotic cells over 1 billion years ago. Eukaryotic cells started out anaerobic and then began to use bacterial machinery- they began to use bacterial oxidative phosphorylation for themselves. This endocytosis occurred when oxygen entered the atmosphere many billions of years ago- this was even before animals and plants separated in the evolutionary tree. Extensive transfer of genes to the eukaryotic nucleus occurred during this process. There is a lot of evidence for this hypothesis, but we can’t prove it happened as we can’t go back in time and observe it happening

Question 45

How were chloroplasts and mitochondria thought to evolve?

Answer 45

Chloroplasts evolved from an ancient form of cyanobacteria and mitochondria evolved from an ancient form of Rickettsia- this is purple photosynthetic bacteria that lost their ability to photosynthesize and were left only with a respiratory chain- therefore, they became mitochondria specialized for oxidative phosphorylation

Question 46

Genetic and structural evidence for the endosymbiont hypothesis (6)

Answer 46

1. Genetic similarity between the DNA found in mitochondrial and chloroplast genes and the DNA found in bacteria. The DNA of these organelles is less similar to the DNA found in the nuclei of the same cell
2. Both of these organelles have inner and outer membranes
3. Whole genomes
4. The DNA of both organelles is arranged like bacteria- some circular genomes, no nucleosome packaging
5. Ribosomes of the organelles are similar to bacterial
6. Cardiolipin is found in the inner membrane of chloroplasts and mitochondria, and of bacteria- it is primarily a bacterial lipid

Question 47

Physiological evidence for the endosymbiont hypothesis (4)

Answer 47

1. Translation starts with N-formyl methionine- this form of methionine is added first in prokaryotic protein synthesis
2. Division similar to binary fission of bacteria- mitochondria and chloroplasts divide by themselves instead of dividing with the cell
3. If these organelles are completely removed, eukaryotic cells cannot produce mitochondria or chloroplasts themselves- does not have the necessary genetic information
4. Enzymes & transport systems similar to bacteria