Transcript 031607

pH and fumarase
Forward reaction: B2 has to accept a proton from water
What if pH is too low?
What if pH is too high?
This week’s lab notes
• You want to know the total activity of each
fraction
slope (Dabs/min) → rate (mmol/min)
Think of this rate as # units of fumarase activity
in the volume you assayed (eg. you may have
added 10 mL to 990 mL assay buffer).
But, you have to correct for the total volume of
the sample. (eg. you may have applied 10.4 mL
of crude to the column)
From Dabs/time
How much of that
sample you tested
for activity (~10mL)
total vol.
Rate 
vol. assayed
Sample
Total
Volume
(mL)
Rate
(mmol/min)
Volume
Assayed
(mL)
Total
Activity
(mmol/min)
Yield
(%)
Crude
10.4
0.25
0.010
260
100
FT
14.1
0.05
0.010
70.5
27.1
Pooled
Elutions
3.2
0.31
0.010
99.2
38.2
Sample’s total activity vs. crude’s
Plan:
• Exam over Ch. 4, 5.1 plus Expt 3 weeks 1
and 2 (fumarase purification and ion
exchange)
• Today: finish up 5.1 (Hb), start Ch. 6
Hemoglobin
• Cooperative binding
– Binding of O2 at one subunit affects the
oxygen affinity of other subunits
• Allostery:
– Regulation by reversible binding at a site
other than the active site
– “Allosteric activation”
– O2: homotropic allosteric activator
Another allosteric modulator
bisphosphoglycerate (BPG)
• Heterotropic allosteric inhibitor
• Binding of Hb•BPG has a lower affinity for
O2 than does Hb
• Enhances release of O2 in the tissues
One BPG molecule per
tetramer
Pushes T ↔ R equilibrium to
the left
T state
High affinity for BPG
Stabilized by BPG
Low affinity for O2
R state
High affinity for O2
Stabilized by O2
Low affinity for BPG
Enzymes
• Biological catalysts
– High specificity and efficiency relative to
inorganic catalysts, for example
– Participate in reactions, but no net change
– Lower the activation energy
– Do not change equilibrium (get there faster)
Enzymes
• Almost exclusively proteins (some RNA,
others?)
• Protein may require cofactor(s)
(non-amino acid functional groups)
– Apoenzyme: protein alone
– Holoenzyme: protein + functional group
– Metals, nucleotide-containing cofactors, etc.
Enzymes
• Usually noted by “-ase” at the end
– DNA polymerase, protein kinase, etc.
• Many enzymes have a common ‘trivial’ name
– Fumarase, hexokinase, lysozyme, etc.
• All enzymes have a systematic name
– Substrate(s) and reaction catalyzed
• Fumarase = “fumarate hydratase”
• Hexokinase = “ATP:glucose phosphotransferase”
Enzymes
• Some common classes of enzymes
– Kinases transfer phosphate (usually from
ATP) to another substrate
– Phosphatases remove (hydrolyze) a
phosphate
– Polymerases string together nucleotides
– Proteases cleave peptide bonds
– Oxidoreductases transfer electrons between
substrates
Drugs often modulate
the action of enzymes
Arachidonic acid
aspirin
Prostaglandin H2
CYCLOOXYGENASE
www.3dchem.com
Enzymes speed up biological reactions
H2CO3 → CO2 + H2O
10,000,000x faster + carbonic anhydrase
Biological reaction:
sugar + oxygen ↔ CO2 + water
High energy “Transition state”
Intermediate between R & P
ENERGY (G°)
Activation energy
EA
Reactants (R)
Kinetic
to
DG <barrier
0
Products (P) reaction
Reaction should be
spontaneous
REACTION PROGRESS
Equil should favor
products
The energy barrier is critical for life
• Potentially deleterious reactions are blocked by EA
– Complex molecule degrading to simpler constituents
nucleotide
DNA
http://encyclopedia.quickseek.com/
http://asm.wku.edu
How do enzymes
speed up reactions?
• Lower the activation energy
• Decrease the energy barrier
2H2O2 → 2H2O + O2
Hydrogen peroxide
Isolated:
EA ~ 86 kJ/mol
In the presence of catalase: EA ~ 1kJ/mol
Binding of substrate to enzyme creates
a new reaction pathway
Without enzyme
With enzyme
EA = DG‡
An enzyme changes EA NOT DG
Affects RATE, not EQUILIBRIUM
http://w3.dwm.ks.edu.tw/
How is EA lowered?
EA = DG‡ = DH - TDS
enthalpy
entropy
•
Enzyme’s ‘goal’ is to reduce DG‡
•
Two ways enzymes can affect DG
– Improve DH
– Improve DS
DG‡ = Gtrans.state – Greactants
Enzymes alter the
free energy of the
transition state
Example: More favorable DH
Charge unfavorable
Unstable transition st.
OH- + A
OH-
A
A
BH+
BH+ -
B + H 2O
Ionic interaction stabilizes
the positive charge
BH
+
AOH
Example: More favorable DS
One molecule
Lower disorder (low S)
Unfavorable entropically
Two molecules
More ‘freedom’
Higher disorder (high S)
Example: More favorable DS
ENZYME
ENZYME
Enzyme/Transition state complex
Enzyme/Reactant COMPLEX
Still a single molecule
Essentially a single molecule
Not much difference entropically
Remember
1. Enzymes lower the energy barrier
2. Decrease EA (DG‡)
3. Provide an environment where:
•
•
Transition state is stabilized (lower enthalpy)
Change of disorder (entropy) is minimized