Quiz 1 Flashcards
What are enzymes
Proteins that catalyze biological reactions without being consumed
Conformation is their defining characteristic
How do enzymes work?
By binding to the substrate and stabilizing the reaction intermediate
Lowering the activation energy required to make a reaction proceed
enzyme +substrate<> enzyme substrate complex<> enzyme + product
Why are enzymes so specific and why do they act only on certain substrates?
Binding takes place in “active site”
Sometimes similarly shaped molecules can fit into cleft– structure determines function
What is an active site?
A cleft in the protein which only fits the substrate
How do we assay (measure) enzyme rate?
Disappearance of reactant
Appearance of product
Must have some reasonably simply way to measure amount of that substance expressed as change in concentration vs time
What are 3 things that affect the rate of any chemical reaction
- Concentration- of reactants and products
- Temperature- higher temp leads to faster rate
- Reaction order- number of substrates
What are 4 factors specific to enzyme-catalyzed reactions
- Concentration of enzyme
- Efficiency of enzyme
- Temperature effects on the efficiency of the enzyme
- Presence of inhibitors
How do we get enzymes
Ideally look for cheap, rich source of enzyme
Factor in degree of purity
What is polyphenoloxidase
Enzyme found in potatoes, which may protect against predators
Oxidizes catechol to orthoquinone
What does orthoquinone do
Breaks down into a series of other products, happen to be colored
Appearance of color suggests a way to measure reaction rate-spectrophotometry
Amount of light absorbed in proportional to the amount of product present
What are inhibitors
How is the effect quantified?
Chemicals that decrease the rate of an enzymatic reaction
BY the percent inhibition:
defined as the drop in enzyme activity in the presence of the inhibitor
(rate without inhibitor) - (rate with inhibitor)/ (rate without inhibitor) times 100
Competitive inhibitor
Similar molecular structure to the substrate, enable them to bind to active site
When bound, the enzyme molecule is unavailable to bind the substrate
What is the rate of reaction with competitive inhibitors
At high substrate concentrations relative to concentration of inhibitor, rate of reaction higher.
Because they outcompete. Lower percent inhibition
What is the hallmark of a competitive inhibitor
A percent inhibition that varies according to substrate concentration
Noncompetitive inhibitor
Do not bind to active site, but reduce the ability of an enzyme to process the substrate
Some may remove a necessary cofactor, other can bind to enzyme and reduce flexibility, others change shape of enzyme
These interactions are affected by enzyme concentration and inhibitor concentration, but not substrate concentration
Percent inhibition remains constant as substrate concentration is varied
What are some important factors in an enzyme-catalyzed reaction
Must form ES complex
Rate depends on concentration of both enzyme and substrate
Rate determined by whichever is limiting (usually enzyme)
Vmax
The maximum rate of reaction
Virtually all enzyme active sites are filled all the time (saturated)
Km
The substrate concentration at which the reaction rate is half maximal
Varies with temperature and pH, or presence of inhibitors
Can be used to measure affinity of enzyme for substrate
The lower the Km, the more likely an enzyme is to be operating at its Vmax
Quinones
Produced by the reaction of PPO, broadly toxic to many insects
How did we the enzymes separated from the potatoes
Homogenization- taking the potato, cutting and blending in distilled water to create a suspension. This ruptures cell membranes. Releases PPO, enzyme cofactors, substrates, and products
- Then centrifuge. Allowing heavy particles to move to the bottom. Supernatant has the enzyme
Catechol
Our buffered substrate (pH is the buffer)
Absorbance units
proportional to the amount of light absorbed at aparticular wavelength, which in turn is proportional to the number of colored molecules present
Initial reaction rate
Slope of the line for first two minutes
Important because this is when we know the exact conditions of the tubes.
How many microliters of enzyme was optimal for the first experiment
60 microliters
In part II of enzymes, what did we examine?
Effect of decreasing enzyme concentration on the rate of reaction. Constant substrate concentration
In part III of enzymes, what did we examine?
Effect of decreasing substrate concentrations with constant enzyme concentration
Part IV enzymes
Running reaction at 0 degrees C, 20 degrees C, 45 degrees C, and 65 degrees C
Part V enzymes
Freezing and thawing vs boiling and letting it cool
Freezing: same reaction rate as control
Boiling: denatured
Part VI and VII enzymes
Competitive and noncompetitive inhibitor
Potassium arsenite reacts with adjacent SH groups in a protein and disrupts disulfide bonds necessary for the tertiary structure of the protein
Potassium arsenite is a good non-competitive inhibitor for all except for 1/8 dilution
Part VII- Para-hydroxybenzoic acid- similar structure to catechol
Competitive inhibitor because % inhibition increased as substrate concentration increased
Part VIII enzymes
Nature of enzyme cofactors
Chelating agents- compounds that tightly bind metal ions. Seeing which ones inhibit the enzyme
PTU reacts with copper and will strip from active site because of high affinity
PTU was in highest inhibitor
Potassium cyanide reacts with copper and iron and would remove both from enzyme
In between
Part IX enzymes
Salt concentrations affect reaction rate. Ions in solution destabilize ionic bonds holding enzyme in conformation
PPo can tolerate between 1.125% and 2.25% salt
33% highest inhibitor
