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4LMS0001 Introduction To Biochemistry

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  • Course Code: 4LMS0001
  • University: University Of Hertfordshire
  • Country: United Kingdom

Question:

1.In just a few sentences explain how the 14C in the labelled glucose and fructose is incorporated into the CO2 released.  (Only an overview, precise details of the pathways are not required.)
 
2.What is the significance of the lack of accumulation of lactate in Experiment 1?

3.What two explanations can you offer for the decrease in O2 uptake and CO2 output in the presence of beta drummin?

4.Explain why the results of Experiment 2 point to some blockage of the glycolytic pathway by beta drummin.

5.Explain why the results of Experiment 3 confirm the findings of Experiment 2.

6.Suggest the probable site of beta drummin inhibition from the data in Table 1, explaining your reasoning.

7.Give reasons why fructose-1,6 bisphosphate should be relatively so high.

8.Postulate why beta drummin is effective as an inhibitor only when added to crude extracts but not when added to purified enzymes. 

9.What is the probable mechanism of inhibition shown when beta drummin acts in vivo?

10.What is the physiological explanation for the anti-fertility effect of beta drummin?
 
 

Answer:

Introduction:

1) According to the Embden-Meyerhof pathway, the pentose cycle involves the conversion of glucose molecules (and fructose molecules) to CO2. Glycolysis involves the conversion of glucose to produce ATP. 3 pentose phosphates elements are converted to form fructose 6-phosphate and triose phosphate . The 14C are used directly while the fructose 6-P is not broken down to Glucose. As such, the 14C is used up to complete for the pentose phosphate cycle due to randomized of 14C metabolism use in the pentose cycle or glycolysis pathway. In some cases, when the fructose-6-phosphate is equal or in equilibrium with glucose-6-phosphate, fructose-6-phosphate can be reversed. However, even when the process is reversed in glycolysis the use of 14C from the substrates. Hence, randomized use of 14C in glucose-6-phosphate is predicted for metabolism in order to complete the pentose cycle.

2) The significance in lack of accumulation of lactate in the experiment is that no abnormalities would be experienced during the pentose cycle. Accumulation in lactate would lead to metabolic dysregulation which would result in reduced or abnormal metabolism of pentose-phosphate . Metabolism would affect the pH balance in pentose-phosphate metabolism if lactate would be produced in high or excess amounts. The other explanation would be the incorporation of the lactate-acid cycle. Since the process is anaerobic the use of CO2 to convert the lactate reduced the amount of carbon (IV) oxide. Nonetheless, as glycolysis is happening probably the lactate acid cycle is taking place. Therefore, based on the results of the experiment, the equilibrium in CO2 and O2 at 50% reduced the production of lactate.

3) The enzymes necessary for metabolic activity may have been depleted or were less compared to the number of available enzymes were lower than substrates . Therefore, the rate of oxygen input in addition carbon (IV) oxide output may have reached an equilibrium. The isomerization of 14C in substrates as a result of reduction of substrates and enzymes necessary for glycolysis. Another reason is that substrates may have reduced in amount which may have resulted in reduced metabolic activity leading to an equilibrium in oxygen input and carbon (IV) oxide output.

4) The opinion is that glycoside beta drummin may be an inhibitor to the enzymatic activity in glycolytic metabolism. Glycolysis heavily relies on enzymatic action which is crucial to the catalysis of the cycle resulting into the creation of carbon (IV) oxide and intake of oxygen. As such, inability of the enzymes to induce metabolism, there was a reduced oxygen intake and carbon (IV) oxide output. The possible explanation is that the binding of the inhibitor, in this case, glycoside beta drummin, to the substrates, at concentration between 10-100mM created an imbalance in the enzyme-substrate complex as a result reducing metabolism. 

5) The addition of inhibition to RSW did not alter any results and thus indicates that glycoside beta drummin, can cause inhibition reaction in the glycolysis process. The plausible explanation is that glycoside beta drummin contains inhibitor molecules that either bind to the substrate-enzyme complex or on the enzymes limiting their biochemical functionality .

 

6) According to the table (Table 1), the probable site of inhibition is during the production of 3-phosphoglycerate. In the glycolysis cycle, enzyme triose phosphate dehydrogenase allocates the hydrogen atom from Phosphate to the reacting agent NAD+ . Based on the table, there is no evidential percentage control compared to products such as glyceraldehyde-3-phosphate which is marked at >300 while 3-phosphoglyceraldehyde is marked as “not-detected.” Therefore, the probable explanation is that the enzyme-substrate complex, triose phosphate dehydrogenase is inhibited to produce glycolysis product 2-glyceraldehyde 3-phosphate which also limits the production of subsequent product, 2-phosphoenolpyruvate.

7) Fructose-1,6-biphosphate is considered to be an intermediary for the high glycolysis sustenance and increases ATP (Adenosine Triphosphate) production. Therefore, cells are programmed to exude high amounts of fructose-1,6-biphosphate as possible in order to ensure that high amounts of ATP (Adenosine Triphosphate) are produced and the pentose cycle is maintained. ATP (Adenosine Triphosphate) is an essential by-product that is necessary in enzyme-mediated processes within the cells including intermediary glycolytic pathways and other pathways. Additionally, another probable reason is because, with the limited intake of oxygen, production of fructose-1,6-biphosphate may be essential in production of ATP as a reservation when the metabolism rates are reducing due to reduced oxygen intake and increased carbon (IV) oxide output .

8) The postulation is that with crude extracts, there is increased competition for inhibition of the metabolic processes compared to when the enzymes are in their pure forms. The proposition is that crude extracts may include additional inhibitory factors in addition to the glycoside beta drummin. The linear effect is that glycoside beta drummin will compete with the other inhibitors to seize the enzyme-substrate complex reducing metabolic rates . In this case, beta drummin may compete for the substrate-binding site of the enzymes with the substrate since the substrate and the inhibitor may bind to the same overlapping sites. Therefore, competitive inhibition is exhibited.

9) The inhibition mechanism exhibited by glycoside beta drummin is competitive inhibition. Competitive inhibitors are determined to be substrate-binding site-specific when it comes to the enzymes. Therefore, in this case, the beta drummin overlaps with the binding sites on the enzymes creating an enzyme-inhibitor complex. The proposition is that the substrate is de-linked or displaced from the enzyme. Hence, for each enzyme there is an inhibitor complex that is formed. The complex may not be as effective since, in the experiments there is evidence of oxygen uptake and carbon (IV) oxide production- meaning that glycoside beta drummin is an a highly competitive inhibitor.

10) The physiological explanation is that glucose or pentose phosphate is linked to the motility and ATP concentration in spermatozoa . The inhibition in metabolism of the glycolytic pathway by the glycoside beta drummin through competitive inhibition limits the production of ATP- which is essential in motility and concentration of ATP in spermatozoa. The consequence is that with reduced amounts of glucose there is reduced motility of spermatozoa.

 

References

9.4. Enzyme inhibition mechanisms [WWW Document], n.d. URL https://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch09s04.html (accessed 3.23.18).

Canto, C., Menzies, K.J., Auwerx, J. NAD+ metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus. Cell Metab. 22, 31–53. 2015.

Chung, J.-J., Shim, S.-H., Everley, R.A., Gygi, S.P., Zhuang, X., Clapham, D.E. Structurally distinct Ca 2+ signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility. Cell 157, 808–822, 2014.

du Plessis, S.S., Agarwal, A., Mohanty, G., Van der Linde, M. Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use? Asian J. Androl. 17, 230, 2015.

Hoff, J., Støren, Ø., Finstad, A., Wang, E., Helgerud, J. Increased blood lactate level deteriorates running economy in world class endurance athletes. J. Strength Cond. Res. 30, 1373–1378, 2016.

Katz, J., Wood, H.G. The use of glucose-C14 for the evaluation of the pathways of glucose metabolism. J. Biol. Chem. 235, 2165–2177, 2010.

Vander Heiden, M.G., DeBerardinis, R.J.  Understanding the intersections between metabolism and cancer biology. Cell 168, 657–669, 2017.

Zhang, C.-S., Hawley, S.A., Zong, Y., Li, M., Wang, Z., Gray, A., Ma, T., Cui, J., Feng, J.-W., Zhu, M. Fructose-1, 6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 548, 112, 2017.

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