Question

1. Another highly regulated enzyme in glycolysis is phosphofructokinase (PFK1). There is evidence from some tumors that PFK1 undergoes a posttranslational proteolysis that yields a truncated functional enzyme (47 kD vs 85 kD) that is insensitive to citrate and ATP. How might this work and why would it be beneficial for a tumor cell?
2. Several isoforms of pyruvate kinase (PK) exist, including PK-M2 which exists mainly in embryonic and adult stem cells. PK-M2 has also been found in many tumor cells, but is in an inactive state. Explain why this seemingly counter-intuitive discovery is actually a benefit for tumor cells.
3. What is the normal function of pyruvate dehydrogenase kinase (PDK)?
4. PDK is more active in cancer cells than regular cells due to the presence of the HIF-1a transcription factor. Why is increased activity of PDK beneficial to cancer cells?

Answer 1

1. Phosphofructokinase is an enzyme that phosphorylates fructose 6 phosphate to fructose 1, 6 bis-phosphate in presence of ATP. This enzyme is an allosteric enzyme that is regulated by ATP and citrate. ATP binds to both active site and regulatory site and this will cause inhibition of enzyme activity. However, if it only binds to ATP (when level of ATP is low), it will cause phosphorylation of fructose 6 phosphate due to ATP hydrolysis. Increase citrate will increase ATP levels as it enters TCA cycle. Thus, high citrate and ATP will inhibit PFK enzyme.

In the case of tumor, the PFK-1 is insensitive to citrate and ATP. Thus, even in presence of citrate and high ATP levels, it will still cause phosphorylation of F-6-P to F-1, 6- BP. This will increase glycolysis. Tumor cells live in oxygen deprived environments and hence increased glycolysis will provide then excess ATP for growth and cell proliferation. There are also more glycolytic intermediates produced which aid in biosynthesis. Enhanced glycolysis causes increased lactic acid production that will protect the tumor cells from damage due to ROS.

2. Pyruvate kinase M2 irreversibly converts PEP to pyruvate during glycolysis. It also leads to production of ATP form ADP and Pi. This enzyme differs from the constitutive PFM1 enzyme in forming dimer will low catalytic efficiency. As a result, PEP is cleared is reduced causing a buildup of glycolytic intermediates. These intermediates are fluxed to biosynthetic pathways for increased proliferation. As tumor cells are highly proliferative, PKM2 has a selective advantage in these cells.

3. Pyruvate dehydrogenase kinase (PDK) will phosphorylate pyruvate dehydrogenase (PD), an enzyme that converts pyruvate to acetyl CoA, at three serine residues. Thus PDK will inactivate PD and thereby inhibit glycolysis. This will cause pyruvate to undergo anaerobic fermentation leading to formation of lactic acid/ethanol.

HIF-1 alpha (hypoxia inducible factor 1 alpha) is activated in tumor cells due to low oxygen concentration that prevails in the tumor. HIF 1 alpha is not degraded in tumor cells and binds to ARNT, activating transcription of target genes. PDK is one of the target genes of HIF 1 alpha signaling. Increase PDK levels will lead to Inhibition of PD. This will inhibit the TCA cycle and ETC due to low production of acetyl CoA. Thus, pyruvate is broken down to lactic acid or ethanol and CO2. HIF 1 alpha also up regulates lactate dehydrogenase. This will regenerate the NAD+ required for glycolysis. Lactic acid protects the tumor cell from ROS (reactive oxygen species) produced in mitochondria, thereby giving protection against oxidative stress. The NAD+ produced will increase glycolysis, providing ATP and glycolytic intermediates for proliferation.