Transcript Enzyme kinetics

Enzymes
Most biological catalysts are proteins,
(some REALLY COOL ONES are folded RNAs)
Catalysts - change rate of reaction without net change of catalyst
Cells make lots of enzymes
Enzymes SPEED up reactions
Enzyme
Nonenzymatic
reaction rate (s-1)
Enzymatic
reaction rate (s-1)
Rate enhancement
Carbonic
anhydrase
1.3 x 10-1
1 x 106
7.7 x 106
Triose phosphate
isomerase
4.3 x 10-6
4300
1 x 109
Staphlococcal
nuclease
1.7 x 10-13
95
5.6 x 1014
Enzymes
Enzyme Action
Figure 1-1
Substrate - acted on by enzyme, high specificity
Enzyme - called ____-ase (protease), ES complex very specific,
small amt needed, remade
Enzymes
Substrate specificity
Types of complementarity between enzyme and substrate:
Geometric
Electronic
Hydrophobic
Hydrophilic
Substrate binding sites undergo conformational change when substrate binds
induced fit
“lock-and-key”
Enzymes
E + S
ES
E + P
G’˚ < 0; favorable
Enzymes
Enzymes affect reaction rates, not equilibria
Catalysts enhance reaction rates by lowering activation energy
Rate is set by activation energy G‡
Higher activation energy --> slower reaction
Overall rate of reaction is determined by step with highest
activation energy --> rate-limiting step
Enzymes
Measure rate of catalytic reaction
Velocity of rxn measured by decrease in [substrate] over time OR
increase in [product] over time
Graph linear at first, then flattens out and no more product is made
Can determine velocity of reaction using early data where graph is linear
Initial Rate (V0) = slope of line
Determine rate
Enzymes
Influences on rate of reaction
1. Amount of enzyme
Initial velocity (V0)  amount of active enzyme
Figure 1-3
From this data can determine amount of enzyme in unknown sample
Example - if 1 µg of pure enzyme has a V0 = 10 nmol of prod / min
THEN a cell extract that yields 20 nmol of prod / min has 2 µg
Enzymes
Influences on rate of reaction
2. Temperature
Enzymes are proteins so they will be denatured at T > 50 - 70 ˚C
most enzymes - increasing the temp up to 45 ˚C usually causes increase in rate
Enzymes have an “optimum temperature range” where
their activity is greatest leading to maximum rate (20 - 40 ˚C)
Most enzymes have an optimum temperature.
A general rule of thumb - a reaction's rate approximately doubles with a
10°C increase in reaction or assay temperature. However, since enzymes are
held together by weak non-covalent bonds, at higher temperatures, the
enzyme catalyzed rate slows down rather than increases.
Enzymes
Influences on rate of reaction
3. pH
Activity influenced by pH - why?
Very high or very low pH cause denaturation/inactivity
Most enzymes operate best at neutral pH
BUT today our enzyme - acid phosphatase - has a pH optimum at 4.5
Calculate pH optimum by assaying the enzyme activity in buffers of different
pH
Plot of enzyme activity Vs. pH is often "bell shaped" since two different
amino acid groups of the enzyme are being titrated to different states of
ionization at the different pH values
One of the two possible ionization states of the amino acid side chain is
effective in enzyme catalysis
Enzymes
Influences on rate of reaction
4. [substrate]
Low [substrate] - active site not sat’d, so enzyme NOT working at full capacity
High [substrate] - no open sites, enzyme working at full capacity (Vmax)
Enzymes
Influences on rate of reaction
4. [substrate]
E + S
Slow step
Rate-limiting
k2
k1
ES
k-1
Vmax achieved when saturate enzyme with substrate
E + P
k-2
Under conditions of defined Temp, pH, ionic strength  Km = Kd
(if the rate of break down of ES to P is slower than the back reaction of
ES dissociating to E + S (in other words, Km = Kd, if k-1 >> k2))
Kd = dissociation constant, [substrate] that yields 1/2 saturation of enz
Km and Kd - affinity
Enzymes
For experimental determination of Km and Vmax do Lineweaver-Burk plot
plot 1/V0 vs. 1/[S]
Straight line --> slope, y-intercept, x-intercept
More accurate determination of Vmax
Enzymes
OUR ENZYME - WHEAT GERM ACID PHOSPHATASE
Lyse open cells to get wheat germ AP - add enzyme extraction buffer which has
detergent NP-40 (what happens here?)
Enzyme extract
AP catalyzes removal of phosphate from macromolecule
Use synthetic substrate, nitrophenol phosphate
Substrate
NITROPHENOL PHOSPHATE
(colorless in alkaline)
AP

Products
NITROPHENOL + PHOSPHATE
(yellow in alkaline)
Enzymes
OUR ENZYME - WHEAT GERM ACID PHOSPHATASE
Exp 1
Make wheat germ extract
Reaction time course with pure AP and wheat germ AP
Nitrophenol standards to create std. curve
Determine V0 of rxn catalyzed by pure AP and wheat germ AP
Estimate amount of AP enzyme in wheat germ
Exp 2
Look at effects of [substrate] on velocity of rxns
Reaction time course with pure AP and varying amounts of substrate
Plot MM and LB curves
Calculate Vmax and Km