L6. Metabolism pt.2 (q13-16)

What are the two categories for nutrient uptake methods?

Passive Transport
Active transport

How does Passive Transport work?

relies on diffusion; requires no energy. Import occurs only if solute is at higher concentration outside cell

How does Active Transport work?

for import of nutrients against a concentration gradient (which requires energy)

What is an examples of passive transport (i.e. no energy needed)?

Simple Diffusion and Facilitated Diffusion

How does Simple diffusion work?

is the movement of molecules like (oxygen, carbon, water, ammonia) from an area of high concentration to an area of low concentration directly through a membrane

How does Facilitated Diffusion work?

selective passive transport using channel proteins for specific molecules (ex. glycerol)

What are examples of active transport (i.e. energy required)?

ion-coupled transport and ABC transport

How does ion-coupled transport work?

uses energy of the transmembrane ion gradient. Ex. (symport if in the same direction and antiport if in opposite, for two solutes transport). In essence, this is a preload scheme: the membrane is first ener­gized by exporting protons (primary event); then, the energy is used for transport (secondary event). (example lactose)

How does ATP-binding cassette (ABC) transport work?

uses ATP hydrolysis to pump a nutrient into the cell after it has bound its corresponding periplasmic binding protein. (example: galactose)

How does the phosphotransferase system (PTS) work?

uses a high-energy phosphate group to chemically modify the solute after it is imported, so it can’t exit.

What is the defining characteristic of autotrophs?

They fix inorganic carbon dioxide (CO2) into organic carbon skeletons (such as glucose). They do not rely on other living organisms for food.

What is the defining characteristic of heterotrophs

cannot fix inorganic carbon; they require complex organic molecules synthesized by other organisms to serve as both their carbon backbone and their energy source.

Define Central metabolism (central carbon metabolism)

is the core network of biochemical pathways that all living cells use to break down nutrients, produce cellular energy, and generate building blocks for growth

Define precursor metabolites

The 13 compounds from which all building blocks can be synthesized / are small intermediate molecules generated during central metabolic pathways.

What are 3 common pathways that comprise central metabolism?

Glycolysis, Pentose phosphate cycle, and TCA cycle.

How does the Glycolysis (Embden-Meyerhof- Parnas pathway) work?

glucose is phosphorylated; for each glucose, yields 2 pyruvate, 2 ATP, and 2 NADH

How does the Pentose phosphate cycle work?

alternate route for converting glucose-6-phosphate to triosephosphates, and production of 2 NADPH

How does the TCA (Krebs/Citric Acid) cycle work?

begins with acetyl coA, which is produced from pyruvate (releasing 1 NADH and 1 CO2) . In 1 cycle, produces 2 CO2, 1 GTP, 3 NADH, 1 FADH but not always performed as a complete cycle

How do autotrophs fix CO2 to make their precursor metabolites?

The overwhelming majority of autotrophs fix carbon dioxide via ribulose bisphosphate carboxylase (RuBisCO)

How does the enzyme ribulose bisphosphate carboxylase (RuBisCO) help in carbon fixation?

catalyzes the addition of carbon dioxide to the five-­ carbon sugar-­ phosphate compound, ribulose bisphosphate. This reaction produces a six-­ carbon intermediate that splits spontaneously, yielding two molecules of 3-­phosphoglycerate.

What cycle is RuBisCO an integral part of?

The Calvin Cycle, which, along with the pathways of central metabolism, is sufficient to synthesize all precursor metabolites from carbon dioxide

What is the problem with the Calvin cycle for carbon fixation?

this remarkable pathway is energetically costly, requiring considerable amounts of ATP and reducing power, as shown in the overall reaction:
6 CO 2 + 12 NADPH + 18 ATP ⇒ C6H12(PO3H2) + 12 NADP+ + 18 ADP + 17 Pi

What is an auxotroph? (textbook definition)

An organism that, due to a mutation, has lost the ability to synthesize a par­tic­u­lar metabolite or nutrient; alternatively, an organism that requires an external source of one or more building blocks.

What is a prototroph? (textbook definition)

An organism that can make all the metabolic building blocks from precursor metabolites

What is a auxotroph? (Dr Chen’s definition)

are mutants of the prototrophs that now have one or more nutritional requirements for the building blocks.

can grow if they are supplied with an intermediate AFTER the blocked step

What is an example of an auxotroph?

a leucine auxotroph now needs to be fed leucine since there is a mutation in the pathway for synthesizing its own

What is a prototroph? (Dr Chen’s Definition)

bacteria that have no nutritional requirements for the building blocks

What are the generalized features of a biosynthetic pathway?

E1 E2 ….
Precursor metabolite → Intermediate a → Intermediate b →….→ Endproduct

Sketch an example of a biosynthetic pathway with “precursor,” 2 “intermediates,” and an “end product”; label the enzymes that catalyze each step.

E1 E2 E3
Precursor → Intermediate a → Inter. b →Inter. c → Endproduct

Referring to your pathway, propose 3 different mutations that would result in an auxotroph.

• Mutation in the gene encoding Enzyme 1:

  • Effect: Inactivates Enzyme 1. The cell cannot convert the Precursor into Intermediate A, halting the entire pathway upstream.

• Mutation in the gene encoding Enzyme 2:

  • Effect: Inactivates Enzyme 2. The cell can produce Intermediate A from the Precursor, but it accumulates Intermediate A without being able to advance it to Intermediate B.

• Mutation in the gene encoding Enzyme 3:

  • Effect: Inactivates Enzyme C. The cell accumulates Intermediate B, but the final catalytic step required to synthesize the End Product is missing.

How might you restore growth to auxotrophs that have your proposed 3 different mutations, from your pathway?

• Universal Rescue (Supplementing the End Product): Adding the final End Product directly to the minimal growth medium bypasses the defective pathway entirely for all three mutants, allowing normal growth.

• Intermediate Rescue (Downstream Supplementation):

  • An Enzyme 1 mutant can sometimes be rescued by supplying Intermediate A or Intermediate B.

  • An Enzyme 2 mutant can be rescued by supplying Intermediate B or the End Product.

  • An Enzyme 3 mutant can only be rescued by supplying the End Product.

  • (provide intermediates past the blocked reaction in order to rescue)

How might your pathway be regulated by feedback inhibition?

Feedback inhibition (allosteric regulation) prevents metabolic waste by adjusting pathway activity based on cellular demand: 

• When the intracellular concentration of the End Product rises beyond what the cell requires for growth, excess End Product molecules bind non-covalently to an allosteric (regulatory) site on Enzyme 1 (typically the enzyme catalyzing the first committed step).

• This binding induces a conformational change that decreases Enzyme 1's affinity for the Precursor, slowing down or temporarily halting flux through the entire pathway until the End Product is consumed.

• (T­hese biosynthetic pathways are governed not by the supply of their starting materials, but by the rate their end products are consumed in polymerization reactions!)