enzyme kinetics Flashcards
(16 cards)
protein studies
- isolate and purify proteins
- visualize proteins
- identify proteins
use/study proteins
enzymes
- protein that acts as a catalyst
- makes reaction more favourable (faster/possible) at physiological conditions
- enzyme specificity is a result of having the right shape to hold the substrates and a catalytic center to facilitate the reaction
initial rate and reaction conditions
- what is optimal temperature, pH and ionic strength
R decreases P increases abd enzyme molecules are lost to denaturation
velocity
is used to get non-attenuated rates ( measured quickly and for a short period of time)
- is the slope of the line, in a closed system it will always slow down as reactant is used up
initial velocity is affected by reactant and enzyme concentration
- rate can be limited by availability of substrate or enzyme (transition from E + S - ES)
- rate can be limited by the time it takes for production of product ( transition from ES - EP - E + P)
michaelis menten equation
- overall reaction speed = time 1 and time 2
- under optimal conditions, and with an excess of substrate, the limitinf step is how fast the enzyme can catalyze ES- E+ P
- through formula minipulation, condition assuptions, experimental data and sheer genius
km = [s]when v0=1/2 vmax
km values to explain alcohol allergy
- defect in aldehyde degydrogenase that results in a build up of acetaldehyde after alcohol consuption
- redness in face, rapid heart rate and breathing, abdominal cramping and hot flashes
ALDH1 VS ALDH2
ALDH1 - CYTOPLASMIS AND HAS HIGH KM
ALDH2 found in mitochondria and has low Km - but its a dominant phenotype
- so ALDH2 is drastically impaired, ALDH1 has a higher Km and acetaldehyde accumulates in the blood causing the flush reaction
manipulation od M-M to simplify analysis
- the lineweaver burk plot uses double reciprocal of rate and concentration to place vmax and km on the X and Y interceots
from theorectical to practical
- Km values can be used ti investigate the affinity of an enzyme for a substrate and compare reaction rates in more complicated (living) systems
- L-B plots can be used to visualize the mechanism of inhibition a compound may exert on an enzyme
- inhibition can be competative and non competative and uncompetative
competative inhibition
- compound reversibly binds the active site and blocks substrate binding
- with competitive inhibators the inhibition can be overcome by having a high [S]
- the inhibator will lower the apparent affinity of the enzyme
- with competative inhibators vmax is the same but the Km increases
methanol and competitive inhibators
- if an enzyme has 2 or more possible substrates one may act as a competative inhibator for the other
uncompetitive inhibition
- a compound binds the ES complex and the slows the transition to EP
- but does not bind E alone
- the transition from ES - EP is slower so vmax is slower
- as the ES complex is affected, but not E alone the equilibrium from E+ S - ES shifts to the right to make the affinity appear higher
uncompetitive inhibition of HIV reverse transcriptase
- HIV therapy is a combination of enzyme inhibators
- currently 30 drugs with 8 targets are used in combinations depending on viral resistance
- one class are non nucleoside reverse transcriptase inhibators
non competative inhibition
- a compound binds E and changes the shape of the active site
- as there are fewer functional E protiens, the overall vmax will be reduced
- E and E + S are both bound, so Km is unchanged
non competative inhibators as next generation drugs
- many drugs discovered are competative inhibators as these are the easiest to study
- if an enzymes normal substrate is known, a compound with a similar shape may also bind the active site
- if other enzymes use the substrate = side effects and toxicity
- non competative inhibators may be more selective
