Biochemistry You have successfully completed your internship rotation with the a
ID: 203401 • Letter: B
Question
Biochemistry
You have successfully completed your internship rotation with the antibiotic group at Methylytransformix, and have moved into their Cancer Therapeutics division.
In many cancers, cells use aerobic glycolysis rather than oxidative phosphorylation as their main energy source. This is known as the Warburg effect, and was first described by Otto Warburg in 1924, who observed that cancer cells have a much higher rate of glycolysis and a lower rate of respiration compared to normal cells. He observed that cancer cells have adapted to low oxygen conditions inside solid tumors by not needing oxidative phosphorylation to generate ATP, and that the cancer cells shut down the mitochondria.
In your position at Methyltransformix, you are studying a gene upregulated in tumor cells, an isoform of PFK2, a bifunctional enzyme that has both kinase and phosphatase activities. Isoforms are genes that express proteins that have a similar structure and function, but have slight variations, giving the proteins unique properties that are often tissue specific. In humans there are four different genes for PFK-2, encoding four different isoforms. The genes are all PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) isoforms. The four isoenzymes of PFK2 include the liver-specific version: PFKFB1, one found in the heart: PFKFB2, one that is expressed in the placenta and is inducible in low oxygen conditions, PFKFB3, and finally PFKFB4 a testes specific isoform, which is also essential to cancer cell survival. In cancerous cells, genes are frequently turned “on” or “off”, changing when and where the gene products are being expressed. This is how the cancer cells are able to form tumors, metastasize and grow in new locations, and to grow quickly.
Figure 1 – Summary of the two catalytic activities of the PFKFB isozymes. Kinase activity of PFKFB genes produces fructose 2,6-Bisphosphate by addition of a phosphate to fructose 6-phosphate. Bisphosphatase activity removes a phosphate from fructose 2,6-bisphosphate to produce fructose 6-phosphate. Figure adapted from Lehninger Principals of Biochemistry, fifith edition.
The adenocarcinoma tumor cells you are studying have been found through a cDNA microarray to have high expression levels of one of the PFK2 isoforms, the gene product of PFKFB3 known as iPFK2. Your supervisor suspects that iPFK2 enzyme has unique properties that may allow cancer cells to change their regulation of glycolysis and gluconeogenesis. Your team is investigating this enzyme as a potential cancer chemotherapy target.
In the lab, you have been working to determine what makes iPFK2 unique by characterizing both the kinase and phosphatase enzyme activity for the enzyme as well as isoforms purified from the liver, heart, skeletal muscle, testes and brain.
Table 1: Comparison of the kinase activity properties of the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:
Kinase:
iPFK2
Placental
Liver
Heart
Skeletal Muscle
Testes
Brain
Vmax (mU/mg)
142
57
61
66
90
90
Fru-6-P Km (uM)
32
16
74
56
85
27
Table 2: Comparison of the bisphosphatase activity properties of the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:
Bisphosphatase:
iPFK2
Placental
Liver
Heart
Skeletal Muscle
Testes
Brain
Vmax (mU/mg)
0.2
45
10
154
22
29
Fru 2,6-BisP Km (uM)
130
0.5
40
0.4
21
70
Table 3: Comparison of the activity ratio of Kinase to bisphosphatase activity for the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:
Activity Ratio
iPFK2
Placental
Liver
Heart
Skeletal Muscle
Testes
Brain
Kinase/bisphosphatase
710
1.3
6.1
0.4
4.1
3.1
Please discuss the following questions:
1. Based on the kinase and bisphosphatase activities listed above for the different enzymes, what differences are observed in the iPFK2 enzyme? Consider both the kinase and bisphosphatase activities, as well as the relationship between the two.
2. What would we expect to happen in the adenocarcinoma cells which have high expression of iPFK2? How would it change carbohydrate metabolism? Be sure to explain your answer. How is this different from the other PFK2 enzymes?
Kinase:
iPFK2
Placental
Liver
Heart
Skeletal Muscle
Testes
Brain
Vmax (mU/mg)
142
57
61
66
90
90
Fru-6-P Km (uM)
32
16
74
56
85
27
Fructose 6-phosphate ATP Kinase activity Bisphosphatase activity ADP Fructose 2,6-bisphosphateExplanation / Answer
1. iPFK2 has a high kinase activity compare to all the other isoforms. It clearly shows that Vmax value is very high so it bring down the Fru-6-P Km to the lower level. At lower concentration of fru-6-P also iPFK2 do the glycolysis.
But Liver, Heart, Muscle have lower Vmax so rate of conversion from fru-6-P to Fru-2,6-P is low. In Testes have
high Vmax but also have high Km so it only operate at high level of Fru-6-P. In Brain some what relate to activity of iPFK2 because in brain glycolysis is the only process to get energy like caner cells.
Bisphosphatase use for gluconogenesis process reverse of glycolysis. iPFK2 Vmax is very low it cleary shows it inhibit the gluconogenesis compare to other isoforms.
2.Adenocarcinoma cells iPFK2 boost the glycolysis by activation Fru2,6 bis phosphate. Fru2,6 bis phosphate activate the PFK1 further to boost up the glycolysis process. In cancer cells glycolysis is the only way to get the energy for cell survival due to inhibition of mitochondrial activity. So it boost the carbohydrate metabolism. Other PFK2 are reversable enzyme both PKK-2 and FBpase2 will be active. But iPFK2 only PFK2 is active.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.