Lecture #15: Glycolysis

Give the name of the enzyme that catalyzes the final reaction in glycolysis, which reaction it catalyzes, and what other high energy molecule is produced in the reaction.

*Pyruvate kinase* is the enzyme that catalyzes the final reaction in glycolysis. It catalyzes the reaction; a phosphate is removed from phosphenolpyruvate to form pyruvate. *ATP* is produced with this reaction, as the phosphate is added to ADP.

Describe the main ways that the activity of phosphofructokinase (PFK) is regulated in muscle and in liver and how its regulation directly affects the regulation of hexokinase. Also, explain how these mechanisms of regulation are adaptive for the cell.

*In muscle tissue:* When ATP levels are high, PFK is more likely to be bound by ATP, which is an allosteric inhibitor of PFK (reduces the binding affinity of fructose-6-phosphate) When ATP levels are low, AMP is more abundant and more likely to be bound to PFK. AMP binding to PFK prevents ATP from inhibiting the reaction velocity of PFK. *In liver tissue:* When blood glucose levels rise (after eating), liver cells will do the first two steps of glycolysis, leading to an increase in the concentration of fructose-6-phosphate. More of this is converted to fructose-2,6-bisphosphate, which is a potent activator of PFK. Thus, this increases the reaction velocity of PFK, driving glycolysis forward. This allows for the liver to utilize as much of the blood glucose as possible. Also, fructose-2,6-bisphosphate will minimize the inhibitory effects of high ATP on PFK.

Name the factors that regulate the activity of pyruvate kinase and the effects each regulator has on the enzyme's activity.

ATP and AMP regulate pyruvate kinase (both L and M isoforms) in a similar way as PFK (allosteric regulation). Pyruvate kinases exists in two isoforms in mammalian cells, where one is found in the muscle and one in the liver. They are regulated slightly differently. The liver (L) isoform of pyruvate kinase is subject to regulation by phosphorylation at particular amino acids that are not found in the muscle (M) isoform. When blood glucose levels are low, L-pyruvate kinase is phosphorylated and becomes less active. When blood glucose levels rise again, the phosphatase dephosphorylates the phosphosite on pyruvate kinase, activating pyruvate kinase. This causes phosphoenolpyruvate to be converted to pyruvate, transferring its energy to ADP to form ATP. This regulation is important when blood glucose levels are low. The liver usually hogs a lot of glucose, but the brain and muscles need it most when blood glucose is low.

For the second part of stage 1 of glycolysis (step 4), name the two products that are produced, and which enzyme is involved in their production. Also indicate which of the two products is actually utilized in stage 2 of glycolysis and how the other product eventually can also enter the third stage of glycolysis, and the name of the enzyme involved.

During step 4, fructose-1,6-bisphosphate is cleaved into two three carbon sugars: dihydroxyacetone phosphate (*DHAP*) and glyceraldehyde-3-phosphate (*GAP*). The enzyme involved in their production is aldolase. DHAP and GAP can isomerize. GAP is the major input to the second stage of glycolysis, (2 GAP per 1 glucose). As GAP is used up, DHAP is converted to GAP to balance the equilibrium.

Give the names of the substrates and products in each of the first two reactions in stage 2 of glycolysis and indicate how energy in the form of electrons or phosphoryl groups is captured in each of these reactions.

First reaction: GAP is phosphorylated to 1,3-bisphosphoglycerate (1,3-BPG); oxidation is coupled with reduction of NAD+ to NADH; capture of carbon energy as high phosphoryl transfer potential Second reaction: transfer of phosphate from 1,3-BPG to ADP to form ATP and 3-phosphoglycerate; first time ATP is truly formed

For each of the two stages of glycolysis, summarize briefly what happens in that stage.

Glycolysis is generally regarded as having an energy investment stage to make fructose-1,6-bisphosphate using two molecules of ATP, then breaking that molecule into two three-carbon molecules of glyceraldehyde-3-phosphate. The second stage is the energy generating stage, where two molecules of glyceraldehyde-3-phosphate are broken down into 2 molecules of pyruvate, capturing energy in ATP and NADH along the way.

Name the two major kinds of fermentation and explain why, in the absence of oxygen, pyruvate undergoes fermentation (why it is necessary when oxygen isn't available).

Lactic acid fermentation and alcohol fermentation are the two major kinds of fermentation. Oxygen is required to accept electrons at the end of the electron transport chain. The energy from the high energy electrons in NADH are harnessed by transfer into the electron transport chain. Therefore, under anaerobic conditions, NADH will build up and have nowhere to transfer its electrons, thus not generating any energy. Glucose is ran through glycolysis to generate pyruvate and a couple of ATP. In order for glycolysis to produce any ATP, NAD+ must be present to accept electrons in step 6. This is the step right before we start getting ATP back out of glycolysis. Fermentation will waste the energy in NADH for the sole purpose of regenerating NAD+ to be used as a reactant in step 6.

For the first part of the stage 1 of glycolysis (steps 1-3) name the two enzymes that are regulated and specify what reactions those two enzymes catalyze (name substrates and products).

Step one consists of glucose entering the cell, whereby *hexokinase* phosphorylates glucose, trapping glucose in the cell. Glucose-6-phosphate inhibits hexokinase. If glucose-6-phosphate is not being utilized in glycolysis or being harnessed to form glycogen, more and more hexokinase will be inhibited, allowing glucose to leave the cell and be used elsewhere in the body. Step three is where fructose-6-phosphate is phosphorylated by phosphofructokinase (*PFK*) to generate fructose-1,6-bisphosphate. This is a commitment step in glycolysis. PFK is highly regulated.