Enzymology in the Workplace

Figure 1: Graph showing how activation energy changes in the presence of enzymes. (Study.com, 2019)

Figure 2: schematic of direct ELISA, where the antigen is represented by the red diamond, antibody is shown by the green Y, enzyme is shown by the purple oval, substrate is shown by yellow sphere, colour change of substrate shown by blue sphere. (Courses.lumenlearning.com, 2019)

Figure 3: Simplified pathway of glycogen metabolism. (Özen, 2007). In the absence of glucose-6-phosphatase, the enzyme responsible for synthesising glucose from glucose-6-phosphate, a partially metabolised version of glycogen, Glucose-6-phosphate may be broken down to ribose-5-phosphate, resulting in an excess of uric acid, hyperuricaemia. Glucose-6-phosphate may undergo glycolysis forming triglycerides, or it may be further metabolised to pyruvate, and be modified to produce fatty acids or cholesterol, all of which result in hyperlipidaemia. This pyruvate may also produce lactate or alanine, resulting in lactic acidaemia or hyperalaninaemia.

Figure 4: Mechanism of 7AAD (Rndsystems.com, 2019)

Figure 5: Creation of a recombinant DNA plasmid (Addgene.org, 2019)

Figure 6: Structure of a detergent. Made using ChemDraw Professional

Figure 7: Detergent mechanism of action. Adapted from (Lichtarowicz, 2019) using ChemDraw Professional.

Figure 8: mechanism of insulin. (Chhabra, 2014)

Week 1: Enzymology in the Workplace

Enzymes are biologically active quaternary proteins which act to lower the activation energy of a reaction, without themselves being used up. Because of this property, enzymes are often used in industry, as they speed up the rate of a reaction, as shown in Figure 1. (Study.com, 2019)^[i]

Figure 1: Graph showing how activation energy changes in the presence of enzymes. (Study.com, 2019)

In cell culture, the enzyme trypsin is used to remove adherent cells from a flask by cleaving peptides on the C-terminal of Lysine or Arginine. (Worthington-biochem.com, 2019)[ii] This releases the cells into a single cell suspension, allowing them to be passaged. Leaving trypsin in for too long will begin to damage the cells as it continues to catalyse. Adding serum and media neutralises trypsin by protease inhibitors. (sigmaaldrich.com, 2019)[iii]

Restriction mapping is used to analyse DNA. It utilises restriction endonucleases, these are enzymes which identify specific base-pair sequences on double stranded DNA. Once the sequence is identified the restriction enzyme cuts the DNA, thus fragmenting it. These fragments are then run on an electrophoresis gel, which gives information about the length and the position of the fragment in the longer DNA chain. (Hendrickson, 2017)[iv]  Scientists can use the sequenced DNA to identify mutations which are linked to diseases. Once these have been identified, methods of diagnosis and treatment can become available to patients. (Murnaghan, 2018)[v]

Enzyme-linked immunosorbent assay (ELISA) is a plate-based technique for the detection and quantification of substances. ELISAs work be having a layer of antigen attached to a surface to which a labelled enzyme is attached. Once the target substance is added, colour begins to develop. The intensity of this colour can be identified using immunophenotyping. (Thermofisher.com, 2019)[vi] Enzymes are used in this assay as they have a high specificity, so will only fluoresce when the correct substrates are present. Shown in Figure 2, (Courses.lumenlearning.com, 2019)[vii]

Figure 2: schematic of direct ELISA, where the antigen is represented by the red diamond, antibody is shown by the green Y, enzyme is shown by the purple oval, substrate is shown by yellow sphere, colour change of substrate shown by blue sphere. (Courses.lumenlearning.com, 2019)

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Week 2: Enzymology in the Workplace

The metabolism of glycogen is affected in many diseases.

Figure 3: Simplified pathway of glycogen metabolism. (Özen, 2007). In the absence of glucose-6-phosphatase, the enzyme responsible for synthesising glucose from glucose-6-phosphate, a partially metabolised version of glycogen, Glucose-6-phosphate may be broken down to ribose-5-phosphate, resulting in an excess of uric acid, hyperuricaemia. Glucose-6-phosphate may undergo glycolysis forming triglycerides, or it may be further metabolised to pyruvate, and be modified to produce fatty acids or cholesterol, all of which result in hyperlipidaemia. This pyruvate may also produce lactate or alanine, resulting in lactic acidaemia or hyperalaninaemia.

Type I, Von Gierke disease, refers to absence of the enzyme glucose-6-phosphatase. Glycogen builds-up in the kidneys, liver, and the small intestines. (King, 2019)^[viii] Thus, the absence primarily results in hypoglycaemia, as well as a myriad of other conditions shown in Figure 3, (Özen, 2007)[ix]. An operation which diverts portal blood from the liver to the vena cava may reduce portal hyperglycaemia and can improve the availability of glucose to the peripheral. Use of this on young children with types I, III, and VI showed marked improvement. (Riddell, 1966)^[x].

Type II, Pompe disease, refers to deficiency of acid alpha-glucosidase (GAA). This leads to the expansion of glycogen-filled lysosomes in tissues, particularly affecting muscle and skeletal tissue. GAA is responsible for cleaving glycosidic bonds in the conversion of glycogen to glucose. (Lim, Li and Raben, 2014)^[xi] The effects of this disease can manifest as fatal hypertrophic cardiomyopathy in infants, or trouble breathing. Enzyme replacement therapy (ERT) enabled some patients to survive without a ventilator, as well as improve motor functions. (Koeberl, Kishnani and Chen, 2007)^[xii]

Type III, Forbes disease, refers to deficiency of 1,6-glucosidase, also known as glycogen debrancher. This results in an excess of abnormal glycogen which is harmful to hepatocytes. During childhood many liver disorders develop, although symptoms usually begin to improve during puberty. Muscle weakness becomes more prominent in adulthood. (Genetics Home Reference, 2019)[xiii]

Type IV, Andersen disease, is deficiency of glycogen-branching enzyme (GBE). This deficiency results in long, unbranched chains forming on the glycogen. These abnormal glycogen molecules are then deposited in many different tissues. Type IV currently has no effective treatment, currently the only option is for those who develop cirrhosis is to undergo a liver transplant. (Anastasopoulou, Avichal and de Lima Corvino, 2017)^[xiv]

Type V, McArdle disease, is the absence of muscle phosphorylase. Consequently, glycogen stores are inaccessible during anaerobic respiration, this causes pain and fatigue during exercise. It can also lead to severe muscle damage. Type V has no effective treatment, but patients can monitor their diet to improve these effects. (Quinlivan, Martinuzzi and Schoser, 2014)^[xv]

Week 4: Enzymology in the Workplace

Deoxyribonucleic acid (DNA) is a molecule which enables organisms to transfer genetic information from one generation to the next. DNA is also used in pharmaceutical research.

7-Aminoactinomycin D (7AAD) is a fluorescent cell stain, it is able to distinguish live and dead cells from one another. 7AAD binds to DNA and fluoresces. Live cells have an impermeable membrane to 7AAD, so the dye is unable to enter the cell. Dead cells have damaged or broken membranes, this allows 7AAD to enter the cell and bind to the DNA, shown in Figure 4. Once the sample is run on a flow cytometer, the live and dead cells in a sample can be counted, giving you total cell numbers, and the percentage of live and dead cells. This allows for the impacts of processes to be investigated. (Rndsystems.com, 2019)[xvi]

Figure 4: Mechanism of 7AAD (Rndsystems.com, 2019)

Polymerase chain reaction (PCR) was developed to amplify the signal of a specific piece of DNA. It allows scientists to identify precise information about the genes which cause diseases. It works by denaturing the double stranded DNA into two individual strands. DNA primers are attached to the template in a process called annealing. Once the primers are in place they can replicate the specific sequence of DNA using the enzyme Taq polymerase. This process is continually repeated until enough of the DNA has been replicated. (Science Learning Hub, 2019)^[xvii]

Plasmids are small circles of DNA found in bacteria. Scientists are able to edit these plasmids by “cutting” and “pasting” desired genes from other organisms into the plasmid, shown in Figure 5. (Addgene.org, 2019)^[xviii] The plasmids can then be reintroduced to bacteria where they will continue to replicate independently of the bacteria’s chromosomes. By including antibiotic resistance, any bacteria not containing the new plasmid are killed using antibiotics. The circular shape of the plasmids allows for an opening to be made without the DNA falling apart. Plasmids have been crucial in the creation of synthetic insulin as well as many other treatments. (Science Learning Hub, 2019)^[xix]

Figure 5: Creation of a recombinant DNA plasmid (Addgene.org, 2019)

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Week 5: Enzymology in the Workplace

In general, detergents are formed of two components – a hydrophilic head, and a hydrophobic tail, Figure 6. The hydrophobic tail is able to dissolve in oils, as many surfactant molecules dissolve into the oil, they hydrophilic heads enable the oil to be cleaved from the surface and hold them in suspension in bundles known as micelles, ready to be washed away, shown in Figure 7 (Lichtarowicz, 2019)[xx]

Figure 6: Structure of a detergent. Made using ChemDraw Professional

There are 3 classifications of detergent; ionic, non-ionic, and zwitterionic detergents.

Figure 7: Detergent mechanism of action. Adapted from (Lichtarowicz, 2019) using ChemDraw Professional.

Ionic detergents contain a head group with is either positively (cationic) or negatively (anionic) charged.

Non-ionic detergents contain uncharged hydrophilic heads. These are suited to breaking lipid-lipid interactions. They are often used in the isolation of biologically active membrane proteins. (Serva.de, 2019)[xxi]

Zwitterionic detergents combine the actions of non-ionic and ionic properties. They carry both positive and negative charges but have a net charge of zero. These lack conductivity and electrophoretic mobility, these don’t bind to ion-exchange resins. However, they are able to break down protein-protein interactions. (Caligur, 2008)[xxii]

Sodium dodecyl sulfate (SDS) is an anionic detergent. It contains a negatively charged sulfate group attached to a 12-carbon chain. It is a very strong and biologically harsh, it has the ability to break inter- and intra-molecular bonds, which destroys their biological action. For this reason, it is commonly used to lyse cells for DNA/RNA extraction. (Brown and Audet, 2008)[xxiii]

Triton x-100 is a non-ionic detergent. It disrupts the plasma membrane inducing autolysis. In gram-positive bacteria, it starts hydrolysis of the peptidoglycan layer. (Johnson, 2013)[xxiv] Triton X-100 is used in cell permeabilisation, in the separation of proteins, and in the characterisation of enzymes. (Serva.de, 2019)[xxv]

3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) is a zwitterionic detergent. CHAPS has a cholesterol-like structure and forms very small micelles, this makes them easy to remove via dialysis. (thermofisher.com, 2019)[xxvi] CHAPS detergent is commonly used in protein solubilisation for isoelectric focusing – separation of molecules by their differences in their isoelectric point. (AG Scientific Blog, 2019)[xxvii]

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Week 7: Enzymology in the Workplace

Type 1 diabetes is a metabolic disorder where the body no longer produces the hormone insulin. Synthetic insulin may be manufactured and taken by patients. (Diabetes UK, 2019)[xxviii] There are several methods of insulin treatment; an insulin pump provides the patient with regular doses of insulin throughout the day, an insulin injection using either a needle and syringe or a pen containing insulin these may contain different strengths of insulin to determine how quickly they act and how long they remain active for, or via inhaler whereby the insulin goes into the lungs and then diffuses into the blood. (NIDDKD, 2019)[xxix] Insulin binds to glycoproteins on the surface of a cell. (Kahn, 1985)[xxx] Once activated, the tyrosine-specific protein kinase generates a signal, resulting in the eventual metabolism of glucose, as shown in Figure 8. (Chhabra, 2014)[xxxi]

Figure 8: mechanism of insulin. (Chhabra, 2014)

Urea cycle disorders (UCD) are the absence of the cofactor producer, or any of the first four enzymes in the urea cycle. The lack of these results in an inability to break-down and remove excess nitrogen, leading to a build-up of ammonia. (NORD, 2019)[xxxii] Currently the only long-term solution is a liver transplant. However, the use of human liver cell transplants has been shown to improve metabolism in the urea cycle, allowing patients to survive until liver transplants are available. (Sokal, 2014)[xxxiii] The treatment consists of several intraportal infusions of donor hepatocytes. Patients are treated with immunosuppressants prior to the infusion to prevent immune response which would attack the transplanted cells. After successful transplantation, the donor cells proliferate, producing the missing enzymes, and starting the urea cycle, allowing for the clearance of built-up ammonia. (Stephenne et al., 2005)^[xxxiv]

Gaucher’s disease which occurs as a result of deficiency of glucocerebrosidase. This enzyme is responsible for the catabolism of the lipid glucocerebroside (GlcCer). This results in deposits of GlcCer in the liver, spleen and nervous system. These deposits affect their normal functions and can lead to hepatosplenomegaly – an enlarged spleen and liver, bone disease, and anaemia. (Lanzkowsky, 2011)[xxxv]

Enzyme replacement therapy (ERT) is now available as a treatment. Fortnightly doses of modified glucocerebrosidase help to prevent the progression of Gaucher’s disease and can often reverse symptoms. However, it does not affect the central nervous system. (Genome.gov, 2012)[xxxvi]

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[ix] Özen, H. (2007). Glycogen storage diseases: New perspectives. World Journal of Gastroenterology, [online] 13(17), p.2541. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146814/ [Accessed 11 Jun. 2019].

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[xii] Koeberl, D., Kishnani, P. and Chen, Y. (2007). Glycogen storage disease types I and II: Treatment updates. Journal of Inherited Metabolic Disease, [online] 30(2), pp.159-164. Available at: https://www.researchgate.net/publication/6496523_Glycogen_storage_disease_types_I_and_II_Treatment_updates [Accessed 11 Jun. 2019].

[xiii] Genetics Home Reference. (2019). Glycogen storage disease type III. [online] Available at: https://ghr.nlm.nih.gov/condition/glycogen-storage-disease-type-iii [Accessed 11 Jun. 2019].

[xiv] Anastasopoulou, C., Avichal, D. and de Lima Corvino, D. (2017). Glycogen Storage Diseases Types I-VII: Background, Pathophysiology, Epidemiology. [online] Emedicine.medscape.com. Available at: https://emedicine.medscape.com/article/1116574-overview [Accessed 11 Jun. 2019].

[xv] Quinlivan, R., Martinuzzi, A. and Schoser, B. (2014). Cochrane Database of Systematic Reviews Pharmacological and nutritional treatment for McArdle disease (Glycogen Storage Disease type V). Cochrane Database of Systematic Reviews, [online] (11). Available at: https://www.cochranelibrary.com/cdsr/doi/10.1002/14651858.CD003458.pub5/epdf/full [Accessed 11 Jun. 2019].

[xvi] Rndsystems.com. (2019). Analysis of Cell Viability using 7-Amino Actinomycin D (7-AAD): R&D Systems. [online] Available at: https://www.rndsystems.com/resources/protocols/analysis-cell-viability-using-7-amino-actinomycin-d-7-aad [Accessed 14 Jun. 2019].

[xvii] Science Learning Hub. (2019). Using PCR in medicine. [online] Available at: https://www.sciencelearn.org.nz/resources/2307-using-pcr-in-medicine [Accessed 14 Jun. 2019].

[xviii] Addgene.org. (2019). Addgene: Molecular Biology Reference. [online] Available at: https://www.addgene.org/mol-bio-reference/#origins [Accessed 14 Jun. 2019].

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[xxiii] Brown, R. and Audet, J. (2008). Current techniques for single-cell lysis. Journal of The Royal Society Interface, [online] 5(suppl_2). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504493/ [Accessed 13 Jun. 2019].

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[xxviii] Diabetes UK. (2019). Insulin and diabetes. [online] Available at: https://www.diabetes.org.uk/guide-to-diabetes/managing-your-diabetes/treating-your-diabetes/insulin [Accessed 20 Jun. 2019].

[xxix] NIDDK – National Institute of Diabetes and Digestive and Kidney Diseases. (2019). Insulin, Medicines, & Other Diabetes Treatments | NIDDK. [online] Available at: https://www.niddk.nih.gov/health-information/diabetes/overview/insulin-medicines-treatments [Accessed 20 Jun. 2019].

[xxx] Kahn, C. (1985). The Molecular Mechanism of Insulin Action. Annual Review of Medicine, 36(1), pp.429-451.

[xxxi] Chhabra, N. (2014). Mechanism of action of Insulin ?. [online] Biochemistry for Medics – Clinical Cases. Available at: http://usmle.biochemistryformedics.com/mechanism-of-action-of-insulin/ [Accessed 20 Jun. 2019].

[xxxii] NORD (National Organization for Rare Disorders). (2019). Urea Cycle Disorders (UCD) – NORD (National Organization for Rare Disorders). [online] Available at: https://rarediseases.org/physician-guide/urea-cycle-disorders/ [Accessed 20 Jun. 2019].

[xxxiii] Sokal, E. (2014). Treating inborn errors of liver metabolism with stem cells: current clinical development. Journal of Inherited Metabolic Disease, [online] 37(4), pp.535-539. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4088990/ [Accessed 20 Jun. 2019].

[xxxiv] Stephenne, X., Najimi, M., Smets, F., Reding, R., de Goyet, J. and Sokal, E. (2005). Cryopreserved Liver Cell Transplantation Controls Ornithine Transcarbamylase Deficient Patient While Awaiting Liver Transplantation. American Journal of Transplantation, [online] 5(8), pp.2058-2061. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1600-6143.2005.00935.x?sid=nlm%3Apubmed [Accessed 20 Jun. 2019].

[xxxv] Lanzkowsky, P. (2011). Hematologic Manifestations of Systemic Illness. Manual of Pediatric Hematology and Oncology, pp.87-122.

[xxxvi] Genome.gov. (2012). About Gaucher Disease. [online] Available at: https://www.genome.gov/Genetic-Disorders/Gaucher-Disease [Accessed 20 Jun. 2019].