W2L1: Enzymes Flashcards
Enzymes
proteins that catalyze reactions; highly specific; speed up reactions by factors of 10^6 or more, without being changed by the reaction; bind ligands transiently; lower activation energy by stabilizing the transition state (orienting substrates, changing reactivity, introducing strain); can also couple endergonic reactions with exergonic ones (ex. ATP); max substrate concentration that a concentration of enzyme can handle
Active Site
specific pocket in an enzyme where substrates/ligands will bind
-ase
at the end of name of an enzyme, suffix appended to the name of the substrate or function
Activation Energy
determines rate of reaction; indicated by height of hill between reactant energy and transition state energy; required to initiate reactions; catalysts lower this by stabilizing transition state (orienting substrates, changing reactivity, introducing strain)
Gibbs Free Energy (delta G)
determines the direction of a reaction, not the rate
Enzyme Regulation
feedback inhibition allosteric interactions competitive inhibition enzyme modifications enzyme levels
Feedback Inhibition
the final product of a pathway binds to a site on the first enzyme, making it no longer active, thus stopping production
Allosteric Regulation
conformation os enzyme changes shape when binding a regulatory molecule, allowing another site to become active or inactive for a substrate; can lead to inhibition or activation of the enzyme
Competitive Inhibitors
block substrates from binding at active site
Enzyme Modification
adding or removing a phosphate can activate or inactivate a protein
Enzyme Levels
level of activity of an enzyme can be affected by rate of enzyme synthesis and rate of enzyme breakdown; transcription and translation can be important in determining regulation; breakdown of proteins also determines concentration of enzymes
Whole Cell Lysate/Homogenate
physical disruption of the cell, breaking of the cell wall resulting in proteins being released; high frequency sound, mild detergent, small hole with high pressure, shear cells with close fitting rotating plunger
Centrifugation
spinning cells separates particles by mass and shape/density; can be used to separate organelles and large protein complexes by repeated spinning at different speeds, removing pellet each time
Column Chromatography
determine what’s in each fraction collected during centrifugation; differentiate by charge, size, or specific interactions
Ion-Exchange Chromatography
separates proteins by charge, using negatively charge beads to hold back positively charged proteins
Gel Filtration Chromatography
separates proteins by size; smaller proteins enter pores in beads, flow through column more slowly
Affinity Chromatography
takes advantage of specific protein interactions; ex. using antibodies to bind a specific protein
Producing Antibodies
inject animal with antigen, collect blood serum, purify antibodies with affinity chromatography (using antigen attached to beads)
Epitope
unique part of an antigen recognized by an antibody
Molecular Biology Uses of Antibodies
immunoblotting (Western blotting) identifies presence of certain proteins in a sample; immunoprecipitation isolates specific proteins and binding partners; immunofluorescence microscopy localizes specific proteins within cells
Polyacrimide Gel Electrophoresis
separates proteins by size using a porous gel; use SDS to coat proteins with negative charge, and then use magnet to make proteins separate; smallest moves the fastest
Immunofluorescence
useful to see where things actually occur in a cell and their arrangement; normal antibody attaches to antigen, then FITC-conjugated antibody attaches to primary antibody
Immune-Precipitation
tag a protein protein that binds to proteins in a cell; release specific antibody for that tagged protein; all proteins binding to the tagged protein will be in the precipitate; then use western blot of mass spectrometry
