ph and fumarase forward reaction: b 2 has to accept a proton from water what if ph is too low? what...
TRANSCRIPT
![Page 1: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/1.jpg)
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?
![Page 2: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/2.jpg)
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)
![Page 3: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/3.jpg)
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
![Page 4: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/4.jpg)
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
![Page 5: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/5.jpg)
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
![Page 6: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/6.jpg)
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
![Page 7: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/7.jpg)
One BPG molecule per tetramer
Pushes T ↔ R equilibrium tothe left
![Page 8: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/8.jpg)
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
![Page 9: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/9.jpg)
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)
![Page 10: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/10.jpg)
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.
![Page 11: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/11.jpg)
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”
![Page 12: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/12.jpg)
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
![Page 13: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/13.jpg)
Drugs often modulate the action of enzymes
CYCLOOXYGENASE
aspirin
www.3dchem.com
Arachidonic acid
Prostaglandin H2
![Page 14: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/14.jpg)
Enzymes speed up biological reactions
H2CO3 → CO2 + H2O
10,000,000x faster + carbonic anhydrase
![Page 15: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/15.jpg)
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)
![Page 16: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/16.jpg)
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
![Page 17: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/17.jpg)
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
![Page 18: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/18.jpg)
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‡
![Page 19: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/19.jpg)
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
![Page 20: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/20.jpg)
-
Example: More favorable H
A B
AOHBH
A BH+
+ H2O
+OH-
+
Charge unfavorableUnstable transition st.
A BH+
Ionic interaction stabilizesthe positive charge
OH-
![Page 21: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/21.jpg)
Example: More favorable S
Two moleculesMore ‘freedom’Higher disorder (high S)
One moleculeLower disorder (low S)Unfavorable entropically
![Page 22: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/22.jpg)
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
![Page 23: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/23.jpg)
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
![Page 24: PH and fumarase Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?](https://reader036.vdocuments.site/reader036/viewer/2022081519/56649ef45503460f94c06b64/html5/thumbnails/24.jpg)