Enzymes are specialized proteins or in some cases RNA molecules which speed up reactions in the cell. Without the presence of enzymes, chemical reactions would occur at a much slower pace - so much so life could not be sustained. Enzymes work by binding to the active site of their particular substrate, lower the reactions activation energy, thereby increasing the rate of reaction. This reaction rate is simply defined as the speed in which the reaction occurs, and is influenced by a number of factors such as temperature, pressure or concentration.
Given an enzyme’s ability to catalyze a reaction, they are vital for cellular processes such as signaling pathways, metabolism, and gene expression. Thus in knowing enzyme activity within the cell, i.e. the moles of substrate converted per unit time, scientists can infer the mechanism behind reactions. Enzyme assays are then standardized laboratory procedures which detect the level of enzymes present in a sample.
So how are enzymes detected? By measuring the disappearance of the substrate, or the appearance of the product. This can be done in a variety of ways, but the most common are done either through colorimetry, fluorescence, or luminescence.
- Fluorometric assays are similar to spectrophotometric assays in that they measure light. However, fluorescence measures light emittance after absorbing light at a different wavelength. So fluorometric assays measure the difference in fluorescence from the substrate to the product to measure the enzyme reaction.
- Luminescence measures the emission of light via chemical reaction. During the enzymatic reactions, light emittance can be used to infer product formation.
Some standard enzyme assays measure the activity of kinase, phosphatase, superoxide dismutase, and Beta-Galactosidase.
Kinases are classed as phosphotransferases as they catalyze the transfer of phosphate groups from high-energy molecules to substrates. This process is known as phosphorylation (where a substrate gains a phosphate), and is a key process of glycolysis, the metabolic pathway common to most cells. Thus, kinases are essential for analyzing metabolic activity in cells.
Directly opposite of kinases are the phosphatases, which removes phosphate groups from a substrate.
Superoxide Dismutase catalyzes the partitioning of the superoxide ( O2- ) into ordinary 02 or H2O2. The superoxide molecule is a by-product of oxygen metabolism and can cause cell damage if not regulated. Thus superoxide dismutase is a major defensive mechanism within cells.
Beta-Galactosidase enzymes catalyze the breakdown of Beta-galactosides into monosaccharides, providing energy and free carbon to the cell.
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Video: Bozeman Science/Youtube