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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 (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