1 / 55

Lecture 17

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. General Acid-Base Catalysis.

dai
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. 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. 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

More Related