Enzymes: Biological Catalysts

Enzymes

• Biological catalysts (biocatalysts) are catalytic proteins.
• Thousands of chemical reactions are sped up by enzymes to make a cell/organism work.
• Enzymes lower the activation energy of a reaction.
• During the process, enzymes are neither altered nor consumed.
• Each enzyme molecule can catalyze a reaction many times, even into the billions.

Applications

• Enzymes are used in industries, analytical settings, and in medicine.
• Industrial applications include detergent enzymes like proteases and lipases.
• Enzymes are used in dairy like rennin for cheese production.
• Xylanase is used as a pulp and paper enzyme, while cellulase and ligninase are used in biofuels.
• Analytical applications accurately measure substance levels, like glucose oxidase measuring glucose in diabetics' blood.
• Medicinal applications include enzymes like DNAses, which break up DNA for cystic fibrosis patients, and tissue plasminogen activator (tPA) for heart attack.

Companies and Commercial Enzymes

• Novozyme supplies 50% of the world's enzyme market.
• Novozyme produces over 100 enzymes for several industries including dairy (animal feeds), brewing, and paper.
• Genencor (DuPont) is the second largest.
• Genencor's industrial enzymes are for food & beverages, animal feeds, water treatments, textiles, biofuels, and biodefense applications.
• Several Enzyme companies exist in Ireland.
  • Megazyme produces diagnostic test kits.
  • Sanofi-Genzyme provides treatments for diseases such as Cerezyme.
  • Trinity Biotech produces diagnostic test kits.

Enzyme Terminology

• Enzymes act on specific molecules called substrates.
• Enzymes are sometimes called a ligand.
• Enzymes are proteins, but the active site is the only small part involved in actual reactions.
• Substrates bind in the active site where reactions take place.
• Substrates become products, but enzymes remain unchanged.

Enzyme Activity

• Substrates fit to the active site through a model of lock–key mechanism or the induced fit shape changes when substrate binds.
• Hexokinase helps breaks down glucose
• The reactioninvolves addition of phosphate group to Glucose + ATP -> Glucose-6 phosphate + ADP
• Glucose binding induces a change in shape of molecule and the addition of phosphate group
• In induced fit, charged amino acid residues can pull and stretch the bonds within a substrate.
• The binding of substrates alters the chemical environment within the catalytic site and slightly alters the site conformation.

Energy and Chemical Reactions

• Substrates (reactants) and products in a chemical reaction possess a particular energy amount.
• The difference between them can be expressed the Gibbs free energy (ΔG).
• When a reaction releases energy it has a negative (ΔG) value, and is said to be exergonic and spontaneous.
• A reaction that requires energy input is endergonic and has a positive (ΔG) value.
• The activation energy (Ea) refers to energy required to start a chemical reaction.
• The lower an (Ea) is in a reaction, the faster that reaction will occur.
• Enzymes lower (Ea) in a given reaction, speeding it up x10^5-10^11.
• Enzymes stabilize the transition state, thus lowering its energy.

Factors Affecting Enzyme Activity

• Most enzymes function best at 37°C and are inactivated at higher temperatures.
• Some enzymes are thermostable, so they work at extremely high temperatures > 95°C.
• Enzymes will denature at certain temperatures.
• Most enzymes function best at a pH of 7, with quite narrow ranges.
• Some enzymes work at lower and higher pH ranges.
  • Pepsin in the stomach functions best at pH 2.
• Biotechnology can be used to create better enzymes.

Substrate Concentration

• A reaction reaches a saturation point when all enzyme molecules are occupied.
• Varying the concentration of the enzyme alters the Vmax.
• The point at which half the active sites are occupied represents the Michaelis constant (Km).
• At the Michaelis constant the velocity is V1/2

Coenzymes

• Most enzymes are proteins, but cofactors or coenzymes are additional molecules that help.
• During an enzyme catalyzed reaction, coenzymes may be modified, then regenerated for another reaction.
• Water soluble vitamins (B-complex e.g. niacin) are frequently used coenzymes in metabolism, making them key to diet.
• Metal ions like Zn and Cu are cofactors used frequently that may be firmly or loosely bound in active sites.

Naming Enzymes

• Enzyme names often end in –ase.
  • Sucrose (invertase) catalyzes sucrose hydrolysis.
• The name often describes its function
  • Oxidases catalyze oxidation reactions.
• Some enzymes, like digestion enzymes, have common names, such as rennin, pepsin and trypsin.
• Some names describe the substrate and function
  • Alcohol dehydrogenase oxidizes ethanol.
• There are six functional classes of enzymes
  • EC 1. Oxidoreductases
  • EC 2. Transferases
  • EC 3. Hydrolases
  • EC 4. Lyases
  • EC 5. Isomerases
  • EC 6. Ligases
• Enzymes are classified by the International Union of Biochemists (I.U.B.) as EC numbers
• Each enzyme has an EC number, or classification number, made up of 4 digits.
  • Example: EC: (2.7.1.1) HEXOKINASE

Activase

• Activase (Alteplase) is an enzyme called tissue plasminogen activator (tPA).
• tPA breaks up blood clots (blockages).
• Blood vessel blockages result in heart attacks and strokes (no. 1 cause of death).
• Activase was the first product (1986) from mammalian cells (CHO) to be approved.
• The FDA approved Activase to treat heart attacks, strokes, and pulmonary embolism.

Pulmozyme

• Cystic fibrosis (CF) is a common genetic disease in Ireland.
• CF is caused by mutation in gene (CFTR protein) involved in Cl- transport.
• CF results in build up of mucus in lungs, leading to chronic chest infections and other problems.
• Pulmozyme (Roche) is a DNase, a group of enzymes that breaks up DNA.
• The treatment reduces inflammation and viscosity of mucus, thus decreasing infection.