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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 (abs/min) → rate (mol/min)
Think of this rate as # units of fumarase activity in the volume you assayed (eg. you may have added 10 L to 990 L 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 abs/time
SampleTotal
Volume(mL)
Rate(mol/min)
VolumeAssayed
(mL)
TotalActivity
(mol/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
How much of thatsample you testedfor activity (~10L)
assayedvol
voltotalRate
.
.
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 tothe left
T state
R state
High affinity for BPGStabilized by BPG
Low affinity for O2
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
CYCLOOXYGENASE
aspirin
www.3dchem.com
Arachidonic acid
Prostaglandin H2
Enzymes speed up biological reactions
H2CO3 → CO2 + H2O
10,000,000x faster + carbonic anhydrase
EN
ER
GY
(G
°)
REACTION PROGRESS
G < 0
Reaction should bespontaneous
Equil should favorproducts
Biological reaction:sugar + oxygen ↔ CO2 + water
Reactants (R)
Activation energy
EA
Kinetic barrier to reaction
High energy “Transition state”Intermediate between R & P
Products (P)
The energy barrier is critical for life
• Potentially deleterious reactions are blocked by EA
– Complex molecule degrading to simpler constituents
http://asm.wku.eduhttp://encyclopedia.quickseek.com/
DNAnucleotide
How do enzymes speed up reactions?
• Lower the activation energy
• Decrease the energy barrier
2H2O2 → 2H2O + O2
Isolated: EA ~ 86 kJ/molIn the presence of catalase: EA ~ 1kJ/mol
Hydrogen peroxide
Binding of substrate to enzyme creates a new reaction pathway
http://w3.dwm.ks.edu.tw/
An enzyme changes EA NOT G
Affects RATE, not EQUILIBRIUM
Without enzyme
With enzyme
EA = G‡
How is EA lowered?
• Enzyme’s ‘goal’ is to reduce G‡
• Two ways enzymes can affect G
– Improve H– Improve S
EA =G‡ = H - TS
G‡ = Gtrans.state – Greactants
Enzymes alter the free energy of the
transition state
enthalpy entropy
-
Example: More favorable H
A B
AOHBH
A BH+
+ H2O
+OH-
+
Charge unfavorableUnstable transition st.
A BH+
Ionic interaction stabilizesthe positive charge
OH-
Example: More favorable S
Two moleculesMore ‘freedom’Higher disorder (high S)
One moleculeLower disorder (low S)Unfavorable entropically
ENZYME
Example: More favorable S
Enzyme/Reactant COMPLEX
Essentially a single molecule
ENZYME
Enzyme/Transition state complex
Still a single molecule
Not much difference entropically
Remember
1. Enzymes lower the energy barrier
2. Decrease EA (G‡)
3. Provide an environment where:
• Transition state is stabilized (lower enthalpy)• Change of disorder (entropy) is minimized
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