1 / 39

Enzymology

Enzymology. Dr. Smita Patel The Department of Biochemistry patelss@umdnj.edu. Oct. 2 Introduction to Enzymes Nov. 7 Enzyme Mechanisms Nov. 9 Enzyme Kinetics Nov. 16 Enzyme Kinetics and Ligand binding. Enzymes All chemical reactions in the cell are catalyzed by enzymes

odette
Download Presentation

Enzymology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Enzymology Dr. Smita Patel The Department of Biochemistry patelss@umdnj.edu Oct. 2 Introduction to Enzymes Nov. 7 Enzyme Mechanisms Nov. 9 Enzyme Kinetics Nov. 16 Enzyme Kinetics and Ligand binding

  2. Enzymes • All chemical reactions in the cell are catalyzed by enzymes • General properties of enzymes • Proteins • Catalyst • Higher reaction rate • Milder reaction conditions • Specificity • Capacity for regulation

  3. ENZYMES ARE PROTEINS 1926 James Sumner crystallized urease from jack bean meal and showed that it was a protein. 1930 John Northrop crystallized pepsin and showed that it was also a protein. He suggested that all enzymes are protein. Awarded Nobel prize in 1946.

  4. Nuclear Mitochondrial Ribosomal Microsomal Lysosomal Cytoplasmic

  5. Enzymes • General properties of enzymes • Proteins • Catalyst • Higher reaction rate • Milder reaction conditions • Specificity • Capacity for regulation

  6. Narrow or broad Chymotrypsin and glucokinase have narrow specificity Alcohol dehydrogenase broad specificity CH3CHO + NADH CH3CH2OH + NAD+ • Active site • substrates bind by noncovalent interactions • Electrostatic: ionic, dipole-dipole • H-bonds • Hydrophobic • Substrate binding is reversible SPECIFICITY

  7. Features of Enzyme Active Sites • The Active site is a small part of the enzyme, a pocket, that is lined with a few catalytic amino acids that participate in binding and catalysis • Active sites are clefts or crevices that usually exclude water (unless it is a reactant). The nonpolar character of the active site enhances the reactivity of any polar residues that contribute to the active site • Substrates are bound to enzymes by many weak interactions; i.e. hydrogen bonds, electrostatic, Van der Waals and hydrophobic interactions

  8. Chymotrypsin • Background • Serine protease (active site Ser residue) • Intestinal digestive enzyme • Synthesized as a zymogen in the pancreases and cleaved to be activated • It is a hydrolase and cleaves peptide bonds specifically at the carboxyl side of an aromatic amino acid side chain Chymotrypsin: R: tyr, trp, or phe Trypsin: R: lys (basic amino acid)

  9. Active Site of Chymotrypsin

  10. Active Sites of Serine Proteases

  11. galactose Glucose binding to glucokinase

  12. Models that explain enzyme specificity • Fischer’s lock and key model • “In order to be able to act chemically on one another, an enzyme and its substrate must fit together like a lock and key.” • Emil Fischer (1852-1919) • Awarded Nobel prize in chemistry, 1902

  13. Fischer’s Lock and Key concept enzyme enzyme substrate Led medicinal chemists to design substrate-like inhibitors as potential drugs enzyme Why does the enzyme not catalyze reactions on smaller molecules?

  14. Hexokinase or Glucokinase fast ATP + glucose ADP + glucose-6-phosphate In the cell, the concentration of H2O is much higher than glucose slow ATP + H2O ADP + phosphate

  15. Induced fit theory Daniel Koshland "the substrate may cause an appreciable change in the three-dimensional relationship of the amino acids at the active site" “hand in a glove” rather than “lock in a key” Specific substrate binding induces a conformational change that strengthens binding and activates the enzyme

  16. Experimental evidence: • Binding of glucose induces a large conformational change • The two lobes swing together by 12 A • Glucose is completely engulfed by the protein active

  17. Enzymes • General properties of enzymes • Proteins • Catalyst • Higher reaction rate • Milder reaction conditions • Specificity • Capacity for regulation

  18. Non-enzymatic rates • How do the rates of enzyme-catalyzed reactions compare to uncatalyzed reactions? • Some enzyme-catalyzed reactions are so slow in the absence of an enzyme catalyst that their half-lives begin to approach the age of the earth itself!

  19. Rate Enhancements (kcat/knon) What is the basis for rate acceleration?

  20. Rate Acceleration by Enzymes Carbonic anhydrase Chymotrypsin 100 molecules/s or half life of 7 millisec Phosphatase 10,000 molecules/s half life of 0.5 millisec knon = 0.14 s-1kcat = 106 s-1kcat /knon = 7.1 x 106 t1/2 = 5 seconds t1/2 = 700 nanoseconds

  21. Brief Review of Thermodynamics • System: Matter within a defined region. • Surroundings: Matter in the rest of the universe. • First Law of Thermodynamics: • The total energy of a system and its surrounding is a constant. DE = EB - EA = Q - W where: Q is the heat absorbed by the system and W is the work done by the system. • The change in the energy of a system is solely dependent on • the initial and final states and not on the path of the transformation.

  22. Brief Review of Thermodynamics • Second Law of Thermodynamics: • A process can occur spontaneously only if the sum of the entropies • of the system and its surroundings increases. • Simply put "energy is required to create order". (DSsystem + DSsurroundings) > 0 for a spontaneous process. • This criterion for spontaneity is difficult to measure since entropy • is not readily measured and the entropy change of both the system • and surrounding must be known.

  23. Brief Review of Thermodynamics • Consequently, additional thermodynamic constants were defined • to facilitate measurements. • The free energy, (G) is derived by combining the first and second • laws. The free energy is the net work done on a system in a reversible process at constant temperature and pressure. • DG = DH - TDS is the change in free energy of a system at constant pressure (P) and temperature (T) • DH is the change in enthalpy (heat content) of the system • DS is the change in entropyof the system. • The properties of the surroundings do not enter into this equation.

  24. Themodynamic Criteria for a Reaction • A reaction is spontaneous only if DG < 0 • A reaction is at equilibrium if DG = 0 • a reaction cannot occurspontaneously if DG > 0 • DG is only dependent on the difference in free energy between the final and initial states and is independent of the path of the reaction. • For example, take the model reaction: A + B <-> C + D • DG = DG° + RT ln [C][D]/[A][B]; at equilibrium DG = 0, so • 0 = DG° + RT ln [C][D]/[A][B] or DG° = -RT ln [C][D]/[A][B]; • Keq = [C][D]/[A][B] it follows that: • DG° = -RT ln Keq (standard free energy change)

  25. Relationship of G° to Keq

  26. Rate Acceleration by Enzymes Carbonic anhydrase Chymotrypsin 100 molecules/s or half life of 7 millisec Phosphatase 10,000 molecules/s half life of 0.5 millisec knon = 0.14 s-1kcat = 106 s-1kcat /knon = 7.1 x 106 t1/2 = 5 seconds t1/2 = 700 nanoseconds

  27. Transition State Theory DG† Free energy reactants DG products Reaction coordinate A-B + C A…B…C A + B-C reactants Transition state products The transition state is the highest point in terms of free energy for a reaction step. G† is the free energy of activation. G† = G (ground state) - G transition state.

  28. Eyring’s Equation Relates the rate of the reaction to the difference in Gibbs free energy between the ground state and transition state. k is rate constant, kB is Boltzmann’s constant, T is temperature, h is Plank’s constant

  29. S‡ G S P Rxn coordinate [S‡] K‡ = [S] K‡ S S‡ – DG‡ RT Derivation of the Eyring equation S is converted to P through the transition state, S‡. Assume that S is in equilibrium with S‡, defined by the equilibrium constant, K‡. DG = –RT ln K (R is the gas constant and T is the absolute temperature) DG ‡ = –RT ln K‡ DG ‡ = –RT ln( [S]‡ / [S] )  [S]‡ = [S] exp (eq. 1)

  30. Transition State Theorem As a reaction proceed from reactants to products there exists a Transition State where the reactants have formed an activated complex that can decay back to the original reactants or it can form the new products. Formation of the activated complex is postulated to be the rate-limiting step in the reaction course.

  31. K‡ k‡ S S‡ P -d[S] dt S and S‡ are in rapid equilibrium, and therefore the rate of the reaction is controlled by the decomposition of S‡ into P. = k‡ [S‡] The transition state is an extremely fleeting species. It’s lifetime is equivalent to the vibrational frequency of the bond that is breaking. Thus, the rate constant, k‡, is equal to the vibrational frequency,u, of a bond. k‡ = u = kBT (eq. 2) (kB is the Boltzmann constant and h is the Planck constant) (u = 6.212  1012 at 25ºC) h

  32. -d[S] -d[S] dt dt – DG‡ – DG‡ – DG‡ RT RT RT substitute eq. 1 & eq. 2 into the rate equation: kBT = k‡ [S‡] = [S] exp h kBT Let the rate constant, k = exp According to transition state theory, then: h kBT if DG‡ k if DG‡ k = k [S], where k = exp h Thus, the rate constant, k, is inversely proportional to DG‡

  33. uncatalyzed reaction Enzyme-catalyzed reaction transition states Fre D ‡ G e ene D ‡ G ES EP rgy S E+S D G D G P E+P progress of reaction progress of reaction Enzymes increase rate by stabilizing the transition state structure

  34. Transition State Stabilization S‡ non-enzymatic P‡ enzymatic DDG‡ G Rxn coordinate From the Eyring equation, calculate that a 10-fold rate enhancement requires a DDG‡ of only 1.36 kcal•mol–1 Typical hydrogen bond energy is ~ 3 kcal•mol–1. Thus, an enzyme can achieve great rate enhancements through preferential interactions with the transition state species.

  35. Enzymes have a high affinity for the transition state structures transition states “I think that enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyzed, that is, the molecular configuration is intermediate between the reacting substances and the products of the reaction” Linus Pauling, 1948 D ‡ G ES EP E+S D G E+P progress of reaction

  36. Transition State Analogue Inhibitors • Preferential binding to the transition state rather than the substrate. • Enzyme should be highly sensitive to inhibitors that mimic the transition state. • Transition state analogues are stable molecules that resemble geometric and/or electronic features of the highly unstable transition state.

  37. Transition State Analogs are Enzyme Inhibitors N OH H HN CO2- O= N R Binds 160x tighter 10,000x tighter Proline racemase Cytidine deaminase OH O= H2N H2N HN HN HN O= O= O= N R N R N R uridine

  38. Enzymes DO NOT Alter the Equilibrium Constant or DG of a Reaction uncatalyzed Lower energy barrier Enzyme catalyzed Free energy A B Reaction coordinate

More Related