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Chapter 15 Enzymatic Catalysis

Chapter 15 Enzymatic Catalysis. Read Chapter 15: Sections 1 & 3 (pp. 496-507, 515-528). Active site of a serine protease. Types of Catalysis. Types of catalytic mechanisms that enzymes employ:. Acid-base catalysis Covalent catalysis Metal ion catalysis Electrostatic catalysis

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Chapter 15 Enzymatic Catalysis

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  1. Chapter 15 Enzymatic Catalysis Read Chapter 15: Sections 1 & 3 (pp. 496-507, 515-528) Active site of a serine protease

  2. Types of Catalysis Types of catalytic mechanisms that enzymes employ: • Acid-base catalysis • Covalent catalysis • Metal ion catalysis • Electrostatic catalysis • Catalysis through proximity and orientation effects • Catalysis by preferential binding of the transition state complex

  3. Mechanism of keto-enol tautomerization Carbanion like High free energy Uncatalyzed slow General acid catalyzed General base catalyzed

  4. Amino Acids in General Acid-Base Catalysis The active site of some enzymes contain amino acid functional group, such as those shown here, can participate in acid-base catalysis From Lehninger Principles of Biochemistry

  5. The X-ray structure of bovine pancreatic RNase S RNase S is the catalytically active form of RNase A • RNase A • Digestive enzyme • Secreted by the pancreas into the small intestine • Functions to hydrolyze RNA to its component nucleotides • RNase A reaction incorporates general acid-base catalysis Non-hydrolyzable substrate analog is bound in the active site

  6. The RNase A mechanism Intermediate RNase A has 2 essential His (His 12 & His 119) His 12 (acting as a base abstracts H+) from 2’-OH group Promotes nucleophilic attack on adjacent P atom His-119 acting as acid protonates the leaving group His 12 (acting as an acid) & His 119 as a general base

  7. Covalent catalysis H2O A B A + B In the presence of covalent catalyst: A B + X: A X + B A + X: + B H2O Serine Proteases are good examples • Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway • The formation of the covalent bond provides chemistry that speeds the reaction

  8. Decarboxylation of acetoacetate Uncatalyzed reaction Catalyzed by primary amines catalyzed reaction

  9. Metal ion catalysis • Nearly 1/3 of all known enzymes require the presence of metal ions for catalysis • (metalloenzymes & metal-activated enzymes) • Metal ions participate in 3 different ways: • Bind substrates to orient them properly • Mediate redox reactions • Electrostatic stabilization or shielding of negative charges

  10. Active site of human carbonic anhydrase Zinc lies at the bottom of the active site cleft Catalytic site is regenerated CO2 + H2O HCO3- + H+

  11. O2- O2- O2- O2- O2- 5 Å O2- 24 Å Thr 56 Glu 131 10 Å Arg 141 Thr 135 4 Å Cu Cu Electrostatic catalysis Charge distribution around the active site of the enzymes…. 1) Seem to be arranged so as to stabilize the transition state of the catalyzed reaction 2) Charge distribution apparently guide polar substrates towards their binding sites Superoxide dismutase (SOD) + SOD +2 2O2- + 2H+O2 + H2O2

  12. Catalysis through proximity & orientation effects A B

  13. Catalysis by preferential transition state binding An enzyme may bind transition state of the reaction it catalyzes with greater affinity than its substrate or products

  14. The Serine Proteases(proteolytic enzymes) Trypsin, chymotrypsin, elastase, thrombin, subtilisin Trypsin, chymotrypsin & elastase together form a potent digestive team All these enzymes catalyze the break down of peptide bond Synthesized by pancreas & secreted into the duodenum • All involve a serine in catalysis - thus the name • Ser is part of a "catalytic triad" of Ser, His, Asp • Serine proteases are homologous, but locations of the three crucial residues differ somewhat • Enzymologists agree, however, to number them always as His-57, Asp-102, Ser-195

  15. Relative positions of the active site residues of 3 unrelated serine proteases

  16. Structure of Chymotrypsin Key active site residues 3 polypeptide chains Pocket in which aromatic side chain of the substrate is bound Disulfide bond & aa residues crucial for catalysis From Lehninger Principles of Biochemistry

  17. Close-up of the active site Oxyanion hole Two of the active site residues are partially visible From Lehninger Principles of Biochemistry

  18. Catalytic mechanism of serine protease (Resembles Transition state)

  19. Serine Protease Mechanism A mixture of covalent and general acid-base catalysis • Asp-102 functions only to orient His-57 • His-57 acts as a general acid and base • Ser-195 forms a covalent bond with peptide to be cleaved • Covalent bond formation turns a trigonal C into a tetrahedral C • The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195

  20. Transition state stabilization in the serine protease • In the tetrahedral intermediate • charged carbonyl O enters the oxyanion hole • hydrogen bonds to the backbone NH groups of Gly 193 & Ser 195 • When S binds to the enzyme • trigonal carbonyl C is conformationally constrained from binding to the oxyanion hole

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