Bioc lecture 2

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Name the molecules the INHIBIT glycolysis:

ATP, citrate, acetyl-coA, fatty acids, ketone bodies. These all represent enough energy. Turns off glycolysis in the tissues that aren't reliant on glucose, so that glucose is saved for RBC, brain etc.

What is glucolysis coordinately regulated with?

Glycogen metabolism, gluconeogenesis, lipid synthesis. Means that don't get opposing pathways happening at the same time.

Name the four ways that glycolysis is regulated.

Feedforward activation
Feedback inhibition
Covalent modification (phosphorylation)
Allosteric regulation

Explain the Pasteur effect

Yeast anaerobically metabolise glucose in closed flask...take the lid off, so induce aerobic metabolism, and glucose consumption goes DOWN! Why: Anaerobic glycolysis produces 2 ATP, aerobic glycolysis produces 32 ATP. The yeast are getting more ATP per glucose molecule, so they consume less glucose. (Remember incr. ATP serves to inhibit PFK and pyruvate kinase)

What is happening when glucose consumption goes down during the pasteur effect?

There's an INCREASE in glucose-6-phosphate and fructose-6-phosphate. There's a DECREASE in all the intermediates from fructose-1,6-bisphosphate down. This shows that Phosphofructo kinase is key regulator of glycolysis. Increased ATP during aerobic glycolysis allosterically INHIBITS PFK so pathway doesn't carry on, and get bounce-back build up of F6P and G6P.

How many regulatory points of glycolysis are there? Name them.

Hexokinase (enzyme that catalyses glucose --> glucose-6-phosphate)
Phosphofructokinase (catalyses fructose-6-phosphate --> fructose-1,6-bisphosphate)
Pyruvate Kinase (catalyses phosphoenol pyruvate --> pyruvate in last step of glycolysis).

Describe how HEXOKINASE regulates glycolysis.

Is an example of feedback/product inhibition. Increased glucose-6-phosphate levels INHIBITS hexokinase. Stops the pathway at the START. (NB that high G6P means high energy state).

Describe how PYRUVATE KINASE regulates glycolysis.

Pyruvate kinase has 4 subunits and comes in THREE isoenzyme types (L, M, A). Inhibitors of PK indicate high energy (eg ATP, acetyl-coA, alanine - which is the transamination pair of pyruvate). Activators of PK are its own substrate (phosphoenol pyruvate), AMP (low energy) and fructose-1,6-bisphosphate (eg of feedforward further upstream, and makes sure that PK is ready and working for when substrates come)

Is pyruvate kinase turned on or off by default?

Off. Because normal physiological concentrations of ATP signal enough energy to turn off pyruvate kinase. This is why feed-forward regulation is necessary to turn PK on.

Describe COVALENT MODIFICATION in regulating glycolysis.

Phosphorylation turns off the LIVER isoenzyme of Pyruvate Kinase (ie phosphorylation inhibits the L form of PK). Glucagon stimulates this phosphorylation because we don't want glycolysis in the liver during fasting (want the liver to pump glucose out into blood, and not to burn it itself!)

The muscle isoenzyme of pyruvate kinase is not phosphorylated to be turned off in the presence of glucagon. Why is this?

In the fasting state you still need ATP to contract muscles, otherwise you wouldn't be able to move! Pyruvate kinase is not turned off in muscle when fasting so that phosphoenol pyruvate can still turn into pyruvate which can still feed into the CAC to produce ATP.

What is the purpose of isoenzymes?

Gives tissue-specific effects for a particular enzyme. Matches the needs for specific tissues.

Describe how PHOSPHOFRUCTO KINASE regulates glycolysis.

Is the MAIN regulatory step in glycolysis. Is a tetramer, but in active form separates into two dimers. INHIBITORS of PFK indicate high energy: ATP, citrate. PFK can either have high affinity or low affinity for its substrate (fructose-6-phosphate). Switches between high affinity (R-state, low ATP, PFK turned on, has michalis-menten hyperbolic curve) or low affinity (T-state, high ATP, sigmoidal curve) depending on ATP concentration. Low ATP means PFK has high affinity. High ATP means PFK has low affinity.

When ATP levels in a cell change, what is the enzyme that detects this the most?

Phosphofructo Kinase. ATP alters PFK's affinity for its substrate (fructose-6-phosphate). If ATP is high, PFK has low affinity for F-6-P and it stops (is in T-state, gets hyperbolic curve). If ATP is low, PFK has high affinity for F-6-P and it increases activity (is in R-state, gets sigmoidal curve like an allosteric enzyme).

Is Phosphofructo Kinase turned on or off by default?

OFF. Because physiological levels of ATP inhibit phosphofructo kinase.

How does citrate inhibit PhosphoFructo Kinase?

Citrate actually acts through the ATP inhibition. Citrate makes ATP bind better to PFK at the allosteric site. When this happens, ATP inhibits PFK better.

Give reaction for F-6-P going to F-1,6-BP...

Fructose-6-Phosphate + ATP --> Fructose-1,6-BisPhosphate + ADP. Phosphofructo Kinase catalyses this. ATP IS THE SUBSTRATE FOR phosphofructo kinase!!!

ATP is the substrate for PFK when it turns F6P into F-1,6-BP. How many binding sites does Phosphofructo Kinase have for its substrate ATP?

TWO. One binding site is the active site (has the higher affinity). The second binding site is an allosteric site (is where ATP binds when its inhibiting PFK)

How do ACTIVATORS of Phosphofructo Kinase work?

They RELIEVE ATP inhibition at the allosteric site. Put PFK into its R-State. AMP, ADP, F-6-P substrate, Fructose-2,6-Bisphosphate.

How does Fructose-2,6-Bisphosphate take ATP off the allosteric site of Phosphofructo Kinase so that it's activated?

There is another Phosphofructo Kinase in the body, which isn't for glycolysis! This one is called PFK-2 and (on the side to usual glycolysis) turns some fructose-6-phosphate --> Fructose-2,6-Bisphosphate. When you make more F6P, you start making more F-2,6-BP too. F-2,6-BP stimulates more usual PFK...makes sure that PFK is ready to deal with the increased F-6-P.

What does F-2,6BP do?

It stimulates PFK!!

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