lecture 17 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Lecture 17 PowerPoint Presentation
Download Presentation
Lecture 17

Loading in 2 Seconds...

play fullscreen
1 / 55
dai

Lecture 17 - PowerPoint PPT Presentation

97 Views
Download Presentation
Lecture 17
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. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Lecture 17 • Exams in Chemistry office with M’Lis. Please show your ID to her to pick up your exam. • Quiz on Friday • Enzyme mechanisms

  2. General Acid-Base Catalysis • General acid catalysis- a process in which partial proton transfer from a Brønstead acid (a species that can donate protons) lowers the free energy of a reaction’s transition state. • General base catalysis - process in which partial proton abstraction by a Brønstead base (a species that can combine with a proton) lowers the free energy of a reaction’s transition state. • General acid-base catalysis-a combination of both.

  3. Figure 15-1a Mechanisms of keto–enol tautomerization.(a) Uncatalyzed. Page 497

  4. Figure 15-1b Mechanisms of keto–enol tautomerization.(b) General acid catalyzed. Page 497

  5. Figure 15-1c Mechanisms of keto–enol tautomerization.(c) General base catalyzed. Page 497

  6. H H d O O O + H+ H2O OR C OR C OR C d d + O H H H H O O - H+ OH C OR C H+ + ROH O General Acid Base Catalysis • Ex. Ester hydrolysis +

  7. General Acid-Base Catalysis • Large number of possible amino acids • Requires that they can accept and donate a proton • Glu, Asp • Lys, His, Arg • Cys, Ser, Thr • Also can include metal cofactors • Example can be observed in carboxypeptidase A (both acid and base catalysis)

  8. R CO2- H-C H N d d C O H-C-R O NH Glu270 C-O- C O General Acid-Base Catalysis • Ex. Carboxypeptidase A Zn plays role of acid (4th ligand is normally H2O, but it is displaced by peptide binding) + Arg145 Glu72 Key aas that holds molecule in place His196 Zn++ HO-Tyr248 His69 Tyr also plays role as 2nd acid catalyst O H d d H + Arg Glu acts as base catalyst to polarize water and form nucleophile

  9. Study of Enzyme Mechanisms • X-ray crystallography-crystallize the molecule with substrate in place and compare to crystal structure of the molecule without the substrate (differences in structure) • For carboxypeptidase A they could show that • Water is expelled by binding of substrate • Arg145 moves 2Å closer to the carboxyl group of the substrate • Glu270 moves 2Å towards the C=O group • Tyr248 moves 12Å towards the amide plane of the peptide • Also able to show what aa surround certain groups-Tyr248 in a hydrophobic pocket.

  10. Study of Enzyme Mechanisms • Check the pH profile of the enzyme. • For carboxypeptidase • The coordination of Zn by His69 and His196 (pK 6.0) • Tyr248 (pK 9.1) Example in book: RNAse (p. 499) 8.5 6.7 Log (Vmax/KM) 6 7 8 9 pH

  11. SugA-SugB-SugC-SugD-SugE-SugF Lysozyme (Strain and Acid Catalysis) • Hen Egg White (HEW) Lysozyme digests bacterial cell walls. • Cleaves (1 4) glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) • Requires about 6 sugars for good recognition.

  12. E O O D O OH : O C OH OR OH SugA-SugB-SugC-SugD-SugE-SugF Lysozyme (Strain and Acid Catalysis) • In theory Asp52 C -O O d H+ O OH C Glu35 • Must distort ring into a flat, planar shape • Supply acid catalysis

  13. Lysozyme (Strain and Acid Catalysis) • In practice

  14. Figure 15-12 Interactions of lysozyme with its substrate. Page 510

  15. Figure 15-11 Chair and half-chair conformations. Page 510

  16. Study of Enzyme Mechanisms • Lysozyme • Only the D ring is strained • Glu35 is in a hydrophobic environment • Asp52 is in a hydrophilic environment • Covalent modification of the active site • Block essential groups • May or may not act at active site • Cd or R-As=O (trivalent arsenic)

  17. The Aspartate Proteases • Pepsin, Renin, HIV protease (AZT targets this) • General acid-base catalysis

  18. R' C O NH R Serine hydrolases: trypsin, chymotrypsin, elastase • Synthesized in pancreas as inactive zymogen (ex. trypsinogen) • Generally operate by "charge relay system" • Asp102, His57, Ser195 conserved in all 3 enzymes. Asp102 His57 Ser195 - COO O 1 H N NH H

  19. Serine hydrolases: trypsin, chymotrypsin, elastase 2 Asp102 His57 Ser195 Rate limiting step for amides H COO O R' N NH C : - O NH R Asp102 His57 Ser195 - COO O R' H N NH C O RNH2

  20. Serine hydrolases: trypsin, chymotrypsin, elastase Asp102 His57 Ser195 - COO O R' H N NH C H O H O Asp102 His57 Ser195 3 H COO O R' Rate limiting step for esters N NH C - O O H

  21. Serine hydrolases: trypsin, chymotrypsin, elastase Asp102 His57 Ser195 - COO O H N NH H R' C O O H

  22. Charge-relay systems • Relay charges between amino acid side chains in order to catalyze the reaction.

  23. Summary: various methods to increase rate • Increase frequency of the correct group in the correct place e.g. proximity effect • Lower EA by specific catalysis -acid-base catalysis, nucleophile or electrophile • Raise energy of reactants (closer to EA) - ring distortion, transition state analog • Provide alternate low EA pathway - covalent catalysis.] • Michaelis Menten • Lineweavear Burk • Eadie Hofstee • Competitive inhibition • Noncompetitive inhibition

  24. Terms to review for enzymes • Cofactor • Coenzyme • Prosthetic group • Holoenzyme • Apoenzyme • Lock and Key • Transition analog model • Induced fit • Active site, binding site, recognition site, catalytic site

  25. Catalytic Mechanisms • Acid-base catalysis • Covalent catalysis • Metal ion catalysis • Proximity and orientation effects (ex. anhydride) • Preferential binding of the transition state complex

  26. General Acid-Base Catalysis • Large number of possible amino acids • Requires that they can accept and donate a proton • Glu, Asp • Lys, His, Arg • Cys, Ser, Thr • Also can include metal cofactors (metal ion catalysis) • Example can be observed in RNAse

  27. Figure 15-2 The pH dependence of V¢max/K¢M in the RNase A–catalyzed hydrolysis of cytidine-2¢,3¢ -cyclic phosphate. Example in book: RNAse (p. 499) Page 499

  28. RNAse mechanism • His12 acts as general base-takes proton from RNA 2’-OH-making a nucleophile which attacks the phosphate group. • His119 acts as a general acid to promote bond scission. • 2’,3’ cyclic intermediate is hydrolyzed through the reverse of the first step-water replaces the leaving group. His12 is the acid, His119 acts as the base Page 499

  29. Covalent catalysis • Rate acceleration through the transient formation of a catalyst-substrate covalent bond. • Example-decarboxylation of acetoacetate by primary amines • Amine nucleophilically attacks carbonyl group of acetoacetate to form a Schiff base (imine bound)

  30. Figure 15-4 The decarboxylation of acetoacetate. uncatalyzed e- sink Page 500 Catalyzed by primary amine

  31. Covalent catalysis • Made up of three stages • The nucleophilic reaction between the catalyst and the substrate to form a covalent bond. • The withdrawal of electrons from the reaction center by the now electrophilic catalyst • The elimination of the catalyst (reverse of 1.) • Nucleophilic catalysis - covalent bond formation is limiting. • Electrophilic catalysis-withdrawal of electrons is rate limiting

  32. Covalent catalysis • Nucleophilicity is related to basicity. Instead of abstracting a proton, nucleophilically attacks to make covalent bond. • Good covalent catalysts must have high nucleophilicity and ability to form a good leaving group. • Polarized groups (highly mobile e-) are good covalent catalysts: imidazole, thiols. • Lys, His, Cys, Asp, Ser • Coenzymes: thiamine pyrophosphate, pyridoxal phosphate.

  33. Covalent Catalysis • Form transient, metastable intermediates that can supply bond energy into the reaction. Examples structures Side chain Chymotrypsin Trypsin Elastase acetylcholinesterase NH O Serine RC-O-CH2-CH COO- (acyl ester) Phosphoglucomutase Alkaline phosphatase Serine O NH -O-P-O-CH2-CH O COO- (phosphoryl ester)

  34. Covalent Catalysis Examples structures Group Papain 3-PGAL-DH NH O Cysteine RC-S-CH2-CH COO- (acyl cysteine) CH Succinate thiokinase Histidine O COO- NH -O-P-N O (phosphoryl imidazole)

  35. Covalent Catalysis Examples structures Group Aldolase Transaldolase R' NH Lysine R-C=N-(CH2)4-CH COO- (Schiff base)

  36. Metal ion catalysis • Almost 1/3 of all enzymes use metal ions for catalytic activity. 2 main types: • Metalloenzymes-have tightly bound metal ions, mmost commonly transition metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+ • Metal-activated enzymes-loosely bind metal ions form solution-usually alkali or alkaline earth metals-Na+, K+, Ca2+

  37. Metal ion catalysis • Three ways for catalysis • Binding to substrates to orient them properly for the reaction • Mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state • Electrostatically stabilizing or shielding negative charges.

  38. Serine Hydrolases (Proteases) • Chymotrypsin, trypsin and elastase. • All have a reactive Ser necessary for activity. • Catalyze the hydrolysis of peptide (amide) bonds. • Chymotrypsin can act as an esterase as well as a protease. • Study of esterase activity provided insights into the catalytic mechanism.

  39. O CH3 O- C O CH3 O NO2 C p-Nitrophenylacetate H2O Chymotrypsin 2H+ -O NO2 + Acetate p-Nitrophenolate

  40. Serine Hydrolases (Proteases) • Reaction takes place in 2 phases • The “burst phase”-fast generation of p-nitrophenolate in stoichiometric amounts with enzyme added • The “steady-state phase”-p-nitrophenolate generated at reduced but constant rate; independent of substrate concentration.

  41. Figure 15-18 Time course of p-nitrophenylacetate hydrolysis as catalyzed by two different concentrations of chymotrypsin. Page 516

  42. O O CH3 CH3 O-Enzyme O- C C O CH3 O NO2 C + Enzyme Chymotrypsin p-Nitrophenylacetate FAST -O NO2 p-Nitrophenolate Acyl-enzyme intermediate H2O SLOW 2H+ + Enzyme Acetate

  43. Chymotrypsin • Follows a ping pong bi bi mechanism. • Rate limiting step for ester hydrolysis is the deacylation step. • Rate limiting step for amide hydrolysis is first step (enzyme acylation).

  44. Identification of catalytic residues CH(CH3)2 (active Ser)-CH2OH O + • Identified catalytically important residues by chemical labeling studies. • Ser195-identified using diisopropylphospho-fluoridate (DIPF) • Irreversible! Diisopropylphospho-fluoridate (DIPF) F-P=O O CH(CH3)2 CH(CH3)2 O DIP-enzyme -P=O (active Ser)-CH2O O CH(CH3)2

  45. Identification of catalytic residues • His57 was identified through affinity labeling • Substrate analog with a reactive group that specifically binds to the active site of the enzyme forms a stable covalent bond with a nearby susceptible group. • Reactive substrate analogs are sometimes called “Trojan horses” of biochemistry. • Affinity labeled groups can be identified by peptide mapping. • For chymotrypsin, they used an analog to Phe.

  46. O Identification of catalytic residues CH2 O NH C CH2Cl S CH3 CH O Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

  47. Figure 15-19 Reaction of TPCK with chymotrypsin to alkylate His 57. Page 517

  48. Homology among enzymes • Bovine chymotrypsin, bovine trypsin and porcine elastase are highly homologous • ~40% identical over ~240 residues. • All enzymes have active Ser and catalytically essential His • X-ray structures closely related. • Asp102 buried in a solvent inaccessible pocket (third enzyme in the “catalytic triad”)

  49. X-ray structures explain differences in substrate specificity • Chymotrypsin - bulky aromatic side chains (Phe, Trp, Tyr) are preferred and fit into a hydrophobic binding pocket located near catalytic residues. • Trypsin - Residue corresponding to chymotrypsin Ser189 is Asp (anionic). The cationic side chains of Arg and Lys can form ion pairs with this residue. • Elastase - Hydrolyzes Ala, Gly and Val rich sequences. The specificity pocket is largely blocked by side chains of Val and a Thr residue that replace Gly residues that line the binding pocket of chymotrypsin and trypsin.

  50. Figure 15-20a X-Ray structure of bovine trypsin.(a) A drawing of the enzyme in complex. Page 518