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Understand that a deficiency or reduced activity of enzymes can cause disease (e.g., GSD Type II, hyperlipidaemia).
Understand how enzymes can be used as therapeutic agents, either directly or in gene therapy-based procedures.
Understand how enzymes can be used in many detection methods (i.e., bioluminescence, chemiluminescence, Western blot) and also in a variety of industrial/commercial/pharmaceutical processes (e.g., drug manufacturing).
Understand that enzymes can be engineered to improve their efficiency.

Q: What is the protein component of an enzyme called?
- A. Holoenzyme
- B. Coenzyme
- C. Apoenzyme
- D. Cofactor
Q: What is a coenzyme?
- A. Protein that helps an enzyme function correctly
- B. A secondary enzyme that helps a primary enzyme to work
- C. Non-protein that non-covalently binds to an enzyme to activate it
- D. Enzyme that inhibits another enzyme

Deficiency or reduced activity of enzymes can cause disease.
Glycogen Storage Diseases (GSD)
- Metabolic disorder involving enzymes regulating glycogen metabolism.
- GSD Type II (Pompe’s disease) caused by deficiency of lysosomal α-glucosidase (accumulation of glycogen in lysosome).
- Muscle weakness and nerve damage leading to heart failure.
- Administer myozyme (replaces defective enzyme).

Enzymes can be used as therapeutic agents, e.g., Stroke.
- Apply an exogenous enzyme to treat the disease process.
- e.g., Stroke - break up blood clots (streptokinase, tissue plasminogen activator [t-PA], urokinase).
Cancer
- Acute leukaemia cells can have an enzyme deficiency where they are unable to synthesize asparagine.
- Obtain Asn from healthy tissue as tumour cell growth needs asparagine.
- Asparaginase limits tumour growth by removing Asn (healthy tissue) available to tumour cells.
  - Asparagine = tumour growth
  - Asparaginase = removes asparagine = cell death
Skin Ulcers
- Break in the skin → underlying tissues can be seen.
- Severe ulcers can have an accumulation of dead tissue which impairs healing.
- Topical application of ointment containing collagenase can be used to promote healing.
Lipoprotein Lipase Deficiency (Hyperlipidaemia)
- Defect in lipoprotein lipase.
- Cannot digest fat from diet.
- Elevated chylomicrons [transport lipids from the digestive tract to the liver].
- Pancreatitis, enlargement of liver and spleen, xanthomas (yellow skin lesions).
- Treatment: limit fat intake and gene therapy.

Direct Treatment
- Replace defective gene with normal gene directly into human.
- Package into virus (use a “no symptoms” virus).
- Glybera approved for the treatment of lipoprotein lipase deficiency – others that are coming onto the market include valoctocogene roxaparvovec (haemophilia) targets FactorVIII (zymogen).
- Concerns:
  - Insert in the wrong place?
  - Not all cells get infected (receptors).
  - Protein overload can cause death.
Ex vivo Treatment
- Take out patient cells - place gene into cells and then put cells back into patient.
- The first successful gene therapy occurred in 1990.
  - Adenosine deaminase deficiency.
  - Purine metabolism (nucleic acid breakdown).
  - Development/maintenance of the immune system.
  - Severe combined immunodeficiency - have no immune system.
  - Gene placed into T lymphocytes.
- The first patient is still healthy.

Bioluminescence
- Bioluminescence - release of light.
- Firefly luciferase (found inside abdomen) transforms luciferin to oxyluciferin + light.
- Used to detect reactions that otherwise couldn’t be visualised (e.g., tumour cells in animals).
Chemiluminescence
- Chemiluminescence → light produced when a chemical reaction is triggered in the presence of a catalyst.
- Luminol.
- Biochemistry: Used to detect proteins, nucleic acids.
- Clinical: Detection of blood.
- The presence of heme-enzyme can also cause the release of light (presence of oxidants and basic compounds).
Immunochemical Methods
- Immunochemical - using antibodies (e.g., Western blot).
- Detects the presence of proteins within a sample allows quantitation.
- Antibody has enzyme conjugated to it.
- The enzyme converts substrate to product and colour or light is released.
- Uses antibodies – perform this in Cell Signalling practical.

Many are ‘engineered’ or designed for commercial/industrial applications.
Food Processing:
- e.g., Glucose oxidase = preservation.
- Rennet from calf stomach = coagulates milk in cheese production.
- Proteases (e.g., trypsin, pepsin, etc.) = meat tenderizers, prevention of cloudiness in beer.
Taste Improvement:
- e.g., Glucose isomerase = increases the sweetness of drinks.
- Naringinase = removal of bitter taste in grapefruit juice.
- Lipase from the pancreas = improvement of cheese aroma.

Paper and Textile Industries
- e.g., Glucose isomerase = added to paper to increase its plasticity.
- Amylase = removal of starch from textiles.
- Proteases (trypsin, pepsin) = tanning agent for leather.
Cosmetics:
- e.g., Proteases = removal of necrotic tissue, acne treatment.

Drug Manufacturing
- Removal of impurities e.g., removal of contaminating proteins in Phyollophorus proteus using papain.
- Drug synthesis e.g., synthesis of simvastatin (acyltransferase – LovD).
- Enantioselective reactions e.g., production of S-citalopram by lipase.
- Engineered to improve usefulness.

Enzymes can be engineered to improve their activity or efficiency.
Recombinant DNA technology → alter amino acid sequence.
Alter kinetic properties, regulation, or enhance substrate specificity/activity, increase stability (e.g., heat, solvent, degradation).
E.g., Penicillin acylase (used to produce the first reagent in penicillin manufacture) → engineered to improve substrate selectivity.

Q: What is bioluminescence?
- A. Metabolism of substrate (luciferase) by enzyme (luciferin) to release light
- B. Metabolism of substrate (oxyluciferin) by enzyme (luciferase) to release light
- C. Metabolism of substrate (luciferin) by enzyme (luciferase) to release light
- D. Metabolism of substrate (luciferase) by enzyme (oxyluciferin) to release light
Q: Which of the following modifications of an enzyme would NOT improve the activity or efficiency of the enzyme?
- A. Alter kinetic properties
- B. Decrease substrate specificity
- C. Increase heat stability
- D. Decrease enzyme degradation

Deficiency or reduced activity of enzymes can cause disease.
Enzymes can be used as therapeutic agents e.g., myozyme for GSD Type II, t-PA in stroke, asparaginase in leukaemia, collagenase for skin ulcers.
Gene therapy - direct and ex vivo.
Enzymes can be used in detection methods e.g., bioluminescence, chemiluminescence, Western blot.
Variety of commercial, industrial and pharmaceutical applications for enzymes e.g., food processing, taste improvement, paper/textile industries, cosmetics, drug synthesis.
Enzymes can be engineered to improve their activity or efficiency.