Proof that Women are Trouble

1. Money = VProblems Proof that Women are Trouble • To find a woman you need Time and Money, therefore: • ”Time is Money” so • Therefore: Woman = Money x Money • ”Money is the root of all Problems” • Therefore: Money = (VProblems)2 Woman = Time x Money Time = Money Woman = (Money)2 Woman = Problems

2. Enzymes - Catalysts of Life • High specificity • Accelerates chemical reactions • Optimal at physiological pH and temperature • Organized in complexes and sequences in biochemical pathways • Conserve and transform chemical energy

3. Specific Substrate (S) Enzyme (E) A → B without enzyme: v ≈ 1 with enzyme: v > 107 • Catalysts: • Can be regulated • Stabilise ”transition state” • Decreases the energy of activation • Do not change the equlibrium

4. Enzymes decrease activation energy Without enzyme With enzyme G G ΔG# without enzyme ΔG# with enzyme S S ΔG ΔG P P • ΔG is the SAME ± enzyme • Enzymes are not consumed in the reaction • Enzymes change the reaction velocity but do • NOT change the chemical equilibrium

5. P≈0

6. P≈0 Formation of es: Removal of es: Vmax Km Derivation of the MM Equation

7. v0 P S3 v0 Initial velocity S2 S1 S Time The Velocity of Enzyme Reactions Depends on Substrate Concentration v: catalytic activity (reaction velocity) Measured as mol/l per time k2 k1 E + S ES E + P k-1

8. k2 k1 E + S ES E + P k-1 v0 mol/time S mol The Michaelis-Menten Equation • If V=½·Vmax s=Km • If k-1 >> k2 • Vmax=k2 ∙ et - Generally:Vmax=kcat ∙ et • kcatis also called “turnover”

9. v0 v = constant • s S Specificity constant The Michaelis-Menten Equation at High and Low Substrate Concentrations • s>>Km v = Vmax • s<<Km

10. k1 k2 k-1 Change unit to s-1 by using the MW of the enzyme Determination of kcat E + S ES E + P v = k2 * [ES] Vmax = kcat * [E]total

11. kcat of Selected Enzymes

12. v0 P S3 v0 Initial velocity S2 S1 S Time Determination of Km and Vmax

13. Lineweaver-Burk Hofstee Wilkinson

14. Michaelis-Menten Hofstee Lineweaver-Burk Wilkinson

15. Kinetic Data for dNK Mutant

16. Hill vs. Michaelis-Menten Michaelis-Menten: Vmax = 4.6 ± 0.5 Km = 16.3 ± 5.0 Hill: Vmax = 3.8 ± 0.1 Km = 11.2 ± 0.9 n = 2.2 ± 0.4 Positive cooperativity

17. Inhibition Kinetics

18. Higher s is needed for v= ½ Vmax Kmis increased by the factor α Competitive Inhibition

19. Uncompetitive Inhibition Both Kmand Vmax are reduced by the factor α’

20. Mixed Inhibition Kmis increased by the factor α/α’and Vmax is reduced by the factor α’

21. Uncompetitive Competitive Both Kmand Vmax are reduced by the factor α’ Kmis increased by the factor α

22. Mixed Noncompetitive Inhibition Is a subtype of mixed inhibition. It represents the rare case where α equals α’. In this situation Km remains unchanged. Kmis increased by the factor α/α’and Vmax is reduced by the factor α’

23. Effect of Inhibitors on Apparent Km and Apparent Vmax

24. ?

25. Slope = n -n log K0.5 Hill Plot A plot of log[v/(V-v)] against log s should give a straight line of slope n and intercept on the vertical axis of -n.logK0.5. The intercept on the horizontal axis is equal to logK0.5.