BCH210 - Lecture 17 - Carbohydrate metabolism

what happens in the anabolic state?

-in the anabolic state, insulin signaling increases glucose utilization

→ increases glucose transport into cells

→ increases the expression and activity of enzymes that use glucose as a substrate

anabolic state

-when the cell has a lot of resources

what happens in the catabolic state?

-in the catabolic state, epinephrine and glucagon signal the release of glucose stores and decrease glucose utilization

catabolic state

-when resources are low

what are the 4 pathways involved in carbohydrate metabolism?

1. glycogen synthesis

2. glycogenolysis

3. glycolysis

4. anaerobic metabolism

glycogen synthesis

-storage of excess glucose as glycogen in liver/muscle cells

-anabolic pathway

glycogenolysis

-breakdown of glycogen to release free glucose

-catabolic pathway

glycolysis

-essential for ATP production from simple monosaccharides (Glc/Fru/Gal) in all cells

-catabolic pathway

anaerobic metabolism

-conversion of pyruvate to lactate in anaerobic conditions (no oxygen)

what are the 2 things that regulate glycolysis?

-glucose (no glucose, its off)

-ATP availability

-they regulate whether glycolysis is turned on or off

glycolysis

-doesn’t require any other organelles, just the cytoplasm

-occurs in the cytoplasm

-cells that don’t have a mitochondria can still use glucose for energy through this process

-its a 10 step catabolic pathway that uses glucose and other simple monosaccharides

-starts with glucose (6 carbons)

-is exergonic (releases energy) under standard and cellular conditions

-glucose is being systematically broken down to two pyruvate molecules, generating ATP and NADH in the process

what are the 2 stages of glycolysis?

1. stage 1 → preparation (steps 1-5) (energy input)

2. stage 2 → (steps 6-10) (energy output)

stage 1 of glycolysis (steps 1-5)

-2 ATP are used to prepare this glucose molecule for breakdown generating 2 GAPs (3-carbons) → glyceraldehyde 3-phosphate (G3P)

stage 2 of glycolysis (steps 6-10)

-produces 2 pyruvates (3Cs), 2 NADHs and 4 ATPs, resulting in a net gain of 2 ATP (since 2 ATPs where used at the start to breakdown glucose, an investment at the start)

-ATP is produced during this by substrate-level phosphorylation

substrate-level phosphorylation

-the phosphate gets added on to ADP to make ATP its coming from a substrate, being transferred from one substrate to ADP to make ATP

what are the 2 fates of the pyruvate after glycolysis?

1. anerobic → lactate

2. aerobic → mitochondria → acetyl CoA or oxaloacetate → CO₂ + NADH

anerobic pathway

don’t have a mitochondria or not a lot of oxygen

aerobic pathway

-when there is a mitochondria and oxygen is available

what 2 enzymes are converted into intermediated of the glycolysis pathway?

1. fructose

2. galactose

what is fructose converted to?

-fructose is converted to DHAP (dihydroxyacetone phosphate) and glyceraldehyde-3-phosphate (G3P or GAP)

-is a ketose

process: fructose-6-phosphate (F6P) → fructose 1,6 bisphosphate → DHAP → 2 G3P (or GAP)

what is galactose converted to?

-galactose is converted to glucose-6-phosphate (G6P)

-is an aldose

process: galactose (D-glucose) → glucose-6-phosphate

how is glycolysis linked?

-each one of these steps are linked together in the cytoplasm, the product of one reaction becomes the substrate in the next reaction coupling the enzymatic reactions together

what carbohydrate is an isomer of glucose?

-galactose (both our aldoses)

which steps of glycolysis are very exergonic and therefore, irreversible and only move in the forward direction?

-steps 1,3, and 10

what do the glycolysis steps being linked adjust?

-they adjust the concs. of the molecules to make them more favourable and in a single directions

total substrates and products of glycolysis?

→ (subtrates) Glucose + 2 NAD+ + 2 ADP + 2 Pi —→ (products) 2 Pyruvate + 2 NADH + 2 ATP +2 H+ + 2 H2O

what is the regulation of step 1 of glycolysis?

-hexokinase or glucokinase can be inhibited by the build-up of its product, glucose 6-phosphate (G6P) (too much of it)

—this step has a large delta G and is essentially irreversible

what is the regulation of step 3 of glycolysis?

-phosphofructokinase-1 (PFK-1) is inhibited by the amount of ATP or citrate present, and activated by the amount of AMP, ADP or Fructose 2,6-bisphosphate

—this step has a large delta G and is essentially irreversible

what is the regulation of step 10 of glycolysis?

-pyruvate kinase is inhibited by phosphorylation and other allosteric regulators

—this step has a large delta G and is essentially irreversible

what steps will you see regulation of glycolysis?

-steps 1,3 and 10 is where you will see regulation of glycolysis

other regulation that can occur in glycolysis

1. the products of the reaction can regulate the enzymatic reaction (by either product binding to the active site and block the substrate from binding or bind to an allosteric site changing the conformation of the enzyme (which inactivates the enzyme) if not being used it will turn off (kind of like feedback regulation

2. regulation found is PTMs and phosphorylation

step 1 of glycolysis - hexokinase

1. glucose is phosphorylated using ATP to G6P

2. hexokinase or glucokinase traps glucose in the cell

→ GLUTs can’t bind to G6P

3. G6P will then be used by the cell to:

• generate energy (glycolysis) (broken down to release energy)

• stored for later (as glycogen in liver or muscle cells)

• used to make new molecules

what is the only cell that can reverse step 1 of glycolysis?

-only liver calls have an enzyme that can reverse this step

glucokinasee

-only works on glucose

hexokinases

-works on hexoses (6 carbon monosaccharides)

how does hexokinase binding occur?

*works almost like a hinge

• ATP and Mg2+ bind to the large domain away from glucose binding in the active sites

• substrates bind in the active site via charged and polar residues

• this binding results in a conformational change where the enzyme closes around the substrates – induced fit model of binding

• C6 hydroxyl then attacks the γ(gamma) phosphate producing G6P

how is hexokinase binding regulated?(3)

1. G6P binds to the active site (inhibits)

2. G6P binds to allosteric site (inhibition) away from the active site

3. insulin increases gene expression (activation) → (increases the total amount of enzyme present)

step 3 of glycolysis - phosphofructokinase (PFK-1)

*continues to activate the hexoses

*this is the 2nd ATP requiring step of glycolysis

1. PFK-1 transfers a phosphate group from ATP to the C1 hydroxyl of F-6P

how is PFK-1 regulated?

• inhibited allosterically by ATP and citrate (bind to allosteric site to turn it off)

• activated allosterically by AMP or ADP and fructose 2,6 bisphosphate

• insulin activates PFK-2 (leads to production of fructose 2,6 bisphosphate), so this enzyme can be activated (this indicates a lot of glucose is available)

• glucagon inactivates PFK-2 (which in turn stops the production of fructose 2,6-bisphosphate, which indicates low glucose, so it wants to turn off this pathway

**since this enzyme has 2 ATP binding sites, the active site and an allosteric site

where does fructose 2,6 bisphosphate come from to activate PFK-1?

-it comes from a side reaction

-this side reaction activates PFK-1, this is a form of feed-forward regulation

F-1,6-BP has been shown to activate Ras, a protein involved in stimulating cell growth. Why is this significant but not surprising?

-because when the cell (metabolite) starts to increase it indicates that, 1. there's lots of glucose available, its being converted to glucose-6-phosphate and its coming through the pathway. Which activates all these other proteins, getting them ready to start using this energy and thee metabolites, using glucose to help the cell to grow, signaling to the cell "this is a high energy state" let's go

step 10 of glycolysis - pyruvate kinase

process:

phosphenolpyruvate —pyruvate kinase→ pyruvate (enol form) → pyruvate (keto form)

how is pyruvate kinase regulated?

• high blood-glucose level → (insulin) activates protein phosphatase (uses water), which removes the phosphate from PPK, which is a key step inactivating that pathway

• low blood-glucose level → (glucagon) activates a PKA which phosphorylates PPK and activates the enzyme

• insulin through dephosphorylation activates it

• fructose 1,6 bisphosphate through allosteric binding activates it (also regulates ras)

• glucagon through phosphorylation ihibits it

• ATP through allosteric binding inhibits it (when you have alot of ATP you don’t need to generate more)

• alanine through allosteric binding inhibits it

• indirectly AMP activates it (its levels)

• indirectly acetyl coA inhibits it (its levels

why is the regulation of pyruvate kinase so important?

-because it is involved in the last step of glycolysis, so it is important for being the final stop to shutting down this entire pathway

why would alanine regulate pyruvate kinase?

-because alanine and pyruvate are very similar in strucuture

→ the only difference is the presence of that amino group

-if you have lots of pyruvate it will be converted to alanine similarly, if you have lots of alanine it will be converted to pyruvate

-glutamate is an amino donor here, so it donates its amino group to pyruvate to generate alanine

what is allosteric regulation dependent on?

-it is dependent on structural changes

→binds to somewhere other than the active site, changing the conformation of the protein and turning the protein off

what is the most effective way of inhibiting glycolysis?

-inhibition of glucose transport (because without glucose, no glycolysis will occur)

what happens when glycolysis is inhibited?

-when glycolysis is inhibited, excess glucose can be stored as glycogen in liver and muscle cells

what controls glycolysis?

-hormonal regulation, energy availability, and glycogen metabolism via enzyme (de)phosphorylation