Transcript Decreases - Bio 5068 - Molecular Cell Biology

Molecular Cell Biology
Kinetics: Enzymology
Cooper
Kinetic analysis
• How cells change over long time periods (development,
long term adaptive changes; hours - years)
• Movement of proteins and membranes within cells dynamics of cellular events (sec - hrs)
– Pulse chase analyses
– Real time imaging: GFP and other fluorophores allow
measurement of trafficking, diffusion, etc. (time-lapse,
fluorescence recovery after photobleaching (FRAP),
etc.)
• Kinetics of molecular interactions, enzyme reactions
(msec - min)
Enzymes are catalysts for chemical reactions in cells
Catalyst (enzyme): increases rate of a reaction
Substrate: molecule on which enzyme acts to form product
S ------> P
enzyme
Free energy of reaction
not changed by enzyme.
For a favored reaction
(ΔG negative), enzyme
accelerates reaction.
Graph:
ΔG* = activation energy
ΔG negative overall for forward reaction
Enzymes as Catalysts
Active Site: Region of the enzyme that does the work. Amino
acid residues in this site assume certain 3D conformation,
which promotes the desired reaction.
What does the Enzyme do to cause catalysis?
•High affinity for substrate in its transition state, facilitating
transition to product
•Increased probability of proper orientation of substrates
•Increased local concentration of substrates
•Has atoms in places that push the reaction forward
•Change hydration sphere of substrates
Phases of Enzyme Reactions
• Transient phase
– Accelerating Velocity
– Short (<1s)
– Formation Enzyme-Substrate
Intermediates
• Steady-state phase
– May Not Occur
– Constant Velocity
– Duration up to Several Minutes
– Little Change Levels of Enzyme
– Small Fraction Substrate Consumed
– Small Levels Product Formed
• Exhaustion phase
– Decreasing Velocity
– Depletion of Substrate
– Accumulation of Product
What Can You Learn from What
Happens at Steady State?
• Turnover number => catalytic efficiency of
enzyme
• Affinity of enzyme for substrates
• Lower bounds for rate constants
• Inhibitors and pH variations to probe active site
• Details of mechanism require transient (presteady state) kinetic analysis
How to Measure Enzyme Activity at Steady State
Need an assay that measures the product of the chemical
reaction. For example...
Enzyme β-galactosidase catalyzes this reaction:
lactose --------------------> glucose + galactose
Measure the amount of glucose or galactose over time.
Trick - use a substrate that produces a reaction product
that absorbs light (creates color). Measure absorbance.
Color-Producing Substrates for β-galactosidase
ONPG = ONP-galactose (ONP = o-nitro-phenol)
ONPG --------------> galactose
+
ONP
(colorless)
(colorless)
(yellow)
X-gal = X-galactose (X = 4-chloro-3-bromo indole)
X-gal ---------------> galactose
+
4-Cl-3-Br-indigo
(colorless)
(colorless)
(deep blue)
Measure absorbance with a spectrophotometer
•Beer’s law - concentration proportional to absorbance
•96-well format instruments
Optimizing assay
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No Enzyme -> No Product
Optimize pH, salt, other buffer conditions
Optimize temperature
Choose set of conditions to be kept
constant
• Amount of enzyme
– Linear range of assay
– More is better
Measure Velocity of Reaction
One Single Experiment at One Substrate Concentration
•Plot product vs time
•Determine rate during initial linear phase
Equilibrium?
Steady-state?
Run the Assay at Different Substrate
Concentrations
Plot initial rate (v0)
vs
Concentration of
Substrate [S]
Michaelis-Menten Plot
• What’s interesting or useful about this
plot?
• Can we use this plot to compare results for
different enzymes or conditions?
Deriving an Equation for the Curve
Consider time zero
•We measure the initial velocity of the reaction)
•No product present: Back reaction is neglible, i.e. no k-2.
The initial velocity, v0, is therefore simply:
v0 = k2[ES]
(k2 often called “kcat” - catalysis rate constant)
Problem - [ES] cannot be
measured
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However...
[S0] (the initial concentration of substrate) is known
[P] (product produced) can be measured
[ETotal] (the amount of enzyme added to the reaction)
is known
• The individual rate equations allow us to solve, using
algebra, for [ES] in terms of these known values
At steady state, d[ES]/dt is zero.
So...
Solving for [ES]...
To simply, let’s define a constant, Km, the
Michaelis constant as...
This simplifies the equation:
But... we don’t know [E].
We do know that the total amount of
Enzyme is the sum of E and ES...
[E0] = [E] + [ES]
thus..
[E] = [E0] − [ES]
Substituting for [E]...
Rearrange to solve for [ES]...
From before, the rate (or velocity) of the reaction is...
Substituting for [ES]...
V, the velocity (rate) of the reaction is...
How does v depend on (vary with) S?
V0
Km is the “Michaelis-Menten constant” - the substrate
concentration at which reaction velocity is half-maximal.
Km = (k-1 + k2)/k1
Typical values? nM to mM
Vmax = k2 [E]total = kcat [E]total
Typical kcat values? 1-1000 per second
Consider three situations...
V0
1. [S] very large, much greater than Km
The enzyme will be saturated with substrate.
[S] + Km = ~ [S], so the rate equation simplifies to...
v0 = Vmax
2. [S] very small, much less than Km
[S] + Km = ~ Km , so the equation simplifies to ...
v0 linearly proportional to [S]
3. [S]=Km
v0 = 50% of Vmax
How Km values affect metabolism
• Glucose + ATP --> glucose-6-P + ADP + H+
• Typical cell [glucose] = 5 mM
• Two enzymes catalyze above reaction
– Hexokinase
• Km (glucose) = 0.1 mM
• Km << [S], so velocity independent of [glucose]
• Reaction is inhibited by product--regulated by product utilization
– Glucokinase
• Km (glucose) = 10 mM
• Km > [S], promotes glucose utilization only when [glucose] is high
• Reaction not inhibited by product--regulated by substrate
availability
Determining Km and Vmax
• Estimate Vmax from asymptote, Km from conc. at Vmax/2
• Curve fitting w/ computer programs, inc Excel
• Visual inspection (Graph paper)
•
Lineweaver-Burke plot and others
Michaelis-Menten equation can be rearranged into
“Lineweaver-Burke” equation
From this graph, visually estimate Km and Vmax.
Regulating enzyme activity
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Allosteric regulation
Reversible covalent modifications
Enzyme availability (synthesis, degradation, localization)
Substrate availability (synthesis, degradation, localization)
Inhibition
– By specific metabolites within the cell
– By drugs, toxins, etc.
– By specific analogues in study of reaction mechanism
Competitive Inhibition
Competitive inhibitor ...
• binds to free enzyme
• prevents simultaneous binding of substrate
- i.e. competes with substrate
• Apparent Km of the substrate is therefore increased
• High substrate concentration:
- substrate overcomes inhibition by mass action
- v0 approaches Vmax (which does not change)
Example of Competitive Inhibition
• EtOH Rx for MeOH poisoning
• Methanol (ingested from solid alcohol, paint strippers,
windshield washer fluid, etc.) is metabolized by alcohol
dehydrogenase to formaldehyde and formic acid. Leads
to metabolic acidosis and optic neuritis (from formate)
that can cause blindness.
• Treatment: Infuse EtOH to keep blood concentration at
100-200 mg/dL (legally intoxicated) for long enough to
excrete the MeOH.
• EtOH serves as a competitive inhibitor. Ethylene glycol
poisoning is treated in the same way.
Noncompetitive Inhibition
• Noncompetitive inhibitor ...
• Binds to a site on the enzyme (E or ES) that
inactivates the enzyme
• Decreases total amount of enzyme
available for catalysis, decreasing Vmax
• Remaining active enzyme molecules are
unaffected, so Km is unchanged
Uncompetitive Inhibition
• Uncompetitive inhibitor...
• Binds specifically to the [ES] complex (and
inactivates it
• Fraction of enzyme inhibited increases as
[S] increases
• So both Km and Vmax are affected
Summary: Types of Inhibitors
• Competitive
– Binds Free Enzyme Only
– Km Increased
• Noncompetitive
– Binds E and ES
– Vmax Decreased
• Uncompetitive
– Binds ES only
– Vmax Decreased
– Km Decreased
Plots to Distinguish Types of Inhibitors
• Competitive
No inhibitor
• Uncompetitive
No inhibitor
• Noncompetitive
No inhibitor
Plots show curves
with no inhibitor
vs. presence of
two different
concentrations of
inhibitor
Reading and Homework for Kinetics
• Alberts (5th edition) pp. 159-166
• Lodish (6th edition) pp. 79-85
• See handout or website for homework