Inaczve and inuences gene transcripzon

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Unformatted text preview: .7 kJ/mol •  C2 carbonyl needed for ring spligng •  ReacZon proceeds through ring ­opened, enediol intermediate •  Dimeric enzyme •  Acts as autocrine moZlity factor (AMF) outside the cell Reminder: hemiacetal formation II. Phsophoglucose Isomerase (PGI): Substrate, Intermediate, Product •  Ring opening leads to C1 carbonyl •  Enediol intermediate resolved to C2 carbonyl H OH H OH HC C C O HO C H OH H C OH OH H C OH C OH HO C H H C H C CH2OPO32- CH2OPO32- II. Phosphoglucose Isomerase (PGI) PDB 1X9I C5 ­OH Zghtly sequestered in the ring ­opened conformer, using residues from both chains. Summary PGI •  Chemical logic: trapping ring ­opened enediol form of glucose •  Pathway logic: moving carbonyl to C2 for later use in ring spligng •  Driving force: highly reversible •  Noteworthy Addi8ons: AcZve site at dimer interface; different funcZons in different compartments. III. Phsophofructokinase-1 (PFK-1) ΔG´° =  ­14.2 kJ/mol •  •  •  •  PhosphorylaZon of new C1 OH Mechanism similar to that for hexokinase First commiled step in glycolysis Highly regulated enzyme (Chpt. 15) III. Phsophofructokinase-I (PFK-1) •  Tetramer (dimer shown) •  Mammalian larger than bacterial (90 kDa vs. 35 kDa / monomer) PDB 1PFK Cataly1c Regulatory III. Phsophofructokinase-I (PFK-1) Tetramer PDB 1PFK Summary PFK-1 •  Chemical logic: coupling ATP hydrolysis to fructose phosphorylaZon •  Pathway logic: priming the system for harvesZng the energy of oxidaZon •  Driving force: ATP hydrolysis •  Noteworthy Addi8ons: Highly regulated; regulatory site at dimer interface; first commiled step in glycolysis; essenZally irreversible in vivo Write a plausible reac1on mechanism IV. Fructose 1,6-Bisphosphate Aldolase ΔG´° = 23.8 kJ/mol •  Reverse of aldol condensaZon, taking advantage of C2 carbonyl •  ΔG in vivo is close to zero •  Two classes of aldolases (both are TIM barrels): Class I (animals and plants) use a Schiff base intermediate Class II (fungi & bacteria) use Zn2+ IV. Aldol Condensation H2O O CH3 -CH 2 C H electrophile OH O C H3C H HO- CH2 carbanion nucleophile H + -OH C H new C-C bond O- O -CH 2 C CH2 H O H C O C CH2 C H resonance stabilized enolate + H2O H IV. Aldolase: Reverse Aldol Condensation CH2OPO32- CH2OPO32- CH2OPO32- C O C O C O- HO C H HO C- H HO C H H C O H C O HB H C OH H C OH H CH2OPO32- :B CH2OPO32- IV. Class I Aldolase IV. Class I Aldolase PDB 1ZAI Note Arg, Lys & Glu binding residues in addition to Schiff base formation IV. Class II Aldolase: Using Zn2+ CH2OPO32- CH2OPO32- C O C O HO C H HO C- H H C O H C O H C OH H C OH H CH2OPO32- :B CH2OPO32- How might Zn2+ be used? HB Summary Aldolase •  Chemical logic: reverse aldol condensaZon; two mechanisms (class I & class II) for stabilizing the enolate intermediate (Schiff base and Zn2+) •  Pathway logic: ring spligng; takes advantage of C2 carbonyl (PGI product, step II) •  Driving force: product removal overcomes unfavorable ΔG´° •  Noteworthy Addi8ons: TIM barrel fold used in apparent convergent evoluZon of class I and class II enzymes V. Triose Phosphate Isomerase (TIM) ΔG´° = 7.5 kJ/mol •  ReacZon proceeds through an enediol intermediate •  Rate is diffusion ­limited (means what?) •  For which the TIM ­barrel fold was named Write a plausible reac1on mechanism V. Triose Phosphate Isomerase (TIM) PDB 2VXN TIM Barrel: 8 ­stranded parallel beta sheet connected by 8 helices Summary TIM •  Chemical logic: stabilizes enediol intermediate •  Pathway logic: coverts 3 ­carbon compounds to the common form used in the next step •  Driving force: highly reversible •  Noteworthy Addi8ons: The original TIM barrel; extremely efficient enzyme Summary: Preparatory Phase (–2 ATP) ΔG´° (kJ/mol)  ­16.7 1.7  ­14.2 23.8 7.5 Why is the overall direction forward?...
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