Wk 9.1 Therapeutic Applications of Enzymes

Learning Goals

• Understand that enzyme deficiencies or reduced activity can cause diseases like Glycogen Storage Disease Type II (GSD Type II) and hyperlipidaemia.

• Understand how enzymes can be used therapeutically, either directly or in gene therapy.

• Understand enzyme applications in detection methods (bioluminescence, chemiluminescence, Western blot) and various industrial, commercial, and pharmaceutical processes (e.g., drug manufacturing).

• Understand that enzymes can be engineered to improve efficiency.

Enzyme Deficiency and Disease

• Deficiency or reduced enzyme activity can cause disease.

Glycogen Storage Diseases (GSD): Metabolic disorders involving enzymes that regulate glycogen metabolism.

• GSD Type II (Pompe’s disease): Caused by a deficiency of lysosomal alpha-glucosidase, leading to glycogen accumulation in lysosomes.

  • Results in muscle weakness and nerve damage, potentially leading to heart failure.

  • Treatment involves administering Myozyme, which replaces the defective enzyme.

    

• Lipoprotein Lipase Deficiency (Hyperlipidaemia):

  • Defect in lipoprotein lipase.

  • Impairs the digestion of fat from the diet, leading to elevated chylomicrons (which transport lipids from the digestive tract to the liver).

  • Symptoms include pancreatitis, enlargement of the liver and spleen, and xanthomas (yellow skin lesions).

  • Management involves limiting fat intake through diet, and potentially gene therapy.

    

Enzymes as Therapeutic Agents

• Enzymes Can Be Used As Therapeutic Agents e.g. Stroke

  • Apply an exogenous enzyme to treat disease process

    • e.g. stroke — break up blood clots (streptokinase, tissue plasminogen activator [t-PA], urokinase)

      

• Enzymes Can Be Used As Therapeutic Agents e.g. Cancer

  • Acute leukaemia cells can have an enzyme deficiency where they are unable to synthesise 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

    

• Enzymes Can Be Used As Therapeutic Agents e.g. Skin Ulcers

  • break in the skin » underlying tissues can be seen

  • Severe ulcers can have an accumulation f dead tissue which impairs healing

  • Topical application of ointment containing collagenase can be used to promote healing

    

Gene Therapy

• Direct Treatment:

  • Replacing a defective gene with a normal gene directly in a human.

  • The gene is packaged into a virus (using a “no symptoms” virus).

  • Glybera was approved for treating lipoprotein lipase deficiency. Other treatments in development include valoctocogene roxaparvovec (for haemophilia), which targets Factor VIII (a zymogen).

  • Potential issues include:

    • The gene inserting in the wrong place.

    • Not all cells get infected (due to receptor limitations).

    • Protein overload causing death.

      • 

• Ex Vivo Treatment: *

  • Removing patient cells, inserting a gene into those cells, and then putting the cells back into the patient.

  • The first successful gene therapy occurred in 1990 for:

    • Adenosine deaminase deficiency, which affects purine metabolism (nucleic acid breakdown).

    • This deficiency impacts the development and maintenance of the immune system, leading to severe combined immunodeficiency (SCID), where individuals have no immune system.

    • The gene was placed into T lymphocytes, and the first patient remained healthy.

  • First patient still healthy

    

Enzymes in Detection Methods

• Bioluminescence: Release of light.

  • Firefly luciferase transforms (found inside abdomen) luciferin to oxyluciferin + light.

    

  • Used to detect reactions that otherwise couldn’t be visualised (e.g., tumour cells in animals).

    

• Chemiluminescence: Light produced when a chemical reaction is triggered in the presence of a catalyst.

  • Luminol is a common example.

  • Used in biochemistry to detect proteins and nucleic acids, and clinically for the detection of blood.

    

  • The presence of a heme-enzyme can also cause the release of light in the presence of oxidants and basic compounds.

    

• Immunochemical Methods: Using antibodies (e.g., Western blot).

  • Detects the presence of proteins within a sample and allows for quantitation.

  • An antibody has an enzyme conjugated to it, which converts a substrate to a product, releasing colour or light.

  • Uses antibodies — perform this in cell signalling practical

    

Commercial Applications

• lots and lots

• Many are ‘engineered’ or designed for commercial/industrial applications

• Food Processing:

  • Glucose oxidase for preservation.

  • Rennet (from calf stomachs) to coagulate milk in cheese production.

  • Proteases (e.g., trypsin, pepsin) as meat tenderizers and to prevent cloudiness in beer.

• Taste Improvement:

  • Glucose isomerase to increase sweetness of drinks.

  • Naringinase to remove the bitter taste in grapefruit juice.

  • Lipase from the pancreas to improve cheese aroma.

Industrial Application

• Paper and Textile Industries:

  • Glucose isomerase added to paper to increase its plasticity.

  • Amylase used to remove starch from textiles.

  • Proteases (trypsin, pepsin) used as tanning agents for leather.

• Cosmetics:

  • Proteases used for the removal of necrotic tissue and acne treatment.

Pharmaceutical Applications

• Drug Manufacturing:

  • Removal of impurities, e.g., removing contaminating proteins in Phyllophorus proteus using papain.

  • Drug synthesis, e.g., synthesis of simvastatin using acyltransferase (LovD).

  • Enantioselective reactions, e.g., production of S-citalopram by lipase.

  • Engineered to improve usefulness

Enzyme Engineering

• Enzymes can be engineered to improve their activity or efficiency using recombinant DNA technology to alter the amino acid sequence.

• This can alter kinetic properties, regulation, enhance substrate specificity/activity, and increase stability (e.g., heat, solvent, degradation).

• Example: Penicillin acylase (used to produce the first reagent in penicillin manufacture) is engineered to improve substrate selectivity.

  

Summary

• 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 can be direct or ex vivo.

• Enzymes are 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.