W2L1: Enzymes

Question 1

Enzymes

Answer 1

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

Question 2

Active Site

Answer 2

specific pocket in an enzyme where substrates/ligands will bind

Question 3

-ase

Answer 3

at the end of name of an enzyme, suffix appended to the name of the substrate or function

Question 4

Activation Energy

Answer 4

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)

Question 5

Gibbs Free Energy (delta G)

Answer 5

determines the direction of a reaction, not the rate

Question 6

Enzyme Regulation

Answer 6

feedback inhibition
allosteric interactions
competitive inhibition
enzyme modifications
enzyme levels

Question 7

Feedback Inhibition

Answer 7

the final product of a pathway binds to a site on the first enzyme, making it no longer active, thus stopping production

Question 8

Allosteric Regulation

Answer 8

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

Question 9

Competitive Inhibitors

Answer 9

block substrates from binding at active site

Question 10

Enzyme Modification

Answer 10

adding or removing a phosphate can activate or inactivate a protein

Question 11

Enzyme Levels

Answer 11

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

Question 12

Whole Cell Lysate/Homogenate

Answer 12

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

Question 13

Centrifugation

Answer 13

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

Question 14

Column Chromatography

Answer 14

determine what’s in each fraction collected during centrifugation; differentiate by charge, size, or specific interactions

Question 15

Ion-Exchange Chromatography

Answer 15

separates proteins by charge, using negatively charge beads to hold back positively charged proteins

Question 16

Gel Filtration Chromatography

Answer 16

separates proteins by size; smaller proteins enter pores in beads, flow through column more slowly

Question 17

Affinity Chromatography

Answer 17

takes advantage of specific protein interactions; ex. using antibodies to bind a specific protein

Question 18

Producing Antibodies

Answer 18

inject animal with antigen, collect blood serum, purify antibodies with affinity chromatography (using antigen attached to beads)

Question 19

Epitope

Answer 19

unique part of an antigen recognized by an antibody

Question 20

Molecular Biology Uses of Antibodies

Answer 20

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

Question 21

Polyacrimide Gel Electrophoresis

Answer 21

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

Question 22

Immunofluorescence

Answer 22

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

Question 23

Immune-Precipitation

Answer 23

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