Biochemistry Lecture 19 - Enzyme Mechanisms PDF

BIOL or CHEM 3361 Biochemistry I Mechanisms of Enzyme Action Reading: Chapter 14 Serine Proteases  Trypsin, chymotrypsin, elastase: digestive enzymes secreted as proenzymes/zymogens  Thrombin: blood clotting cascade  Subtilisin: bacterial enzyme  Plasmin: cleaves fibrin...

BIOL or CHEM 3361 Biochemistry I Mechanisms of Enzyme Action Reading: Chapter 14 Serine Proteases  Trypsin, chymotrypsin, elastase: digestive enzymes secreted as proenzymes/zymogens  Thrombin: blood clotting cascade  Subtilisin: bacterial enzyme  Plasmin: cleaves fibrin polymers of blood clots  Tissue plasminogen activator: cleaves plasmin proenzyme plasminogen and is administered to prevent heart attack  Acetylcholinesterase: NOT a protease but mechanistically similar in breaking down acetylcholine Trypsin, Chymotrypsin and Elastase Proenzymes or zymogens All cleave polypeptide chains Similar sequences, structures and mechanisms Colored dot is an aa that is identical Gold lines are disulfide bonds Structure Structure Structure of chymotrypsin (white) in a complex with eglin C (blue ribbon structure), a target substrate. His57 (red) is flanked by Asp102 (gold) and Ser195 (green). The catalytic site is filled by a peptide segment of eglin. Note how close Ser195 is to the peptide that would be cleaved in the reaction. The Catalytic Triad of the Serine Proteases The catalytic triad at the active site of chymotrypsin (and the other serine proteases.) Trypsin, Chymotrypsin and Elastase Similar structures and mechanism, but different specificities Trypsin – carbonyl side of Arg or Lys (basic) EXCEPT WHEN EITHER IS FOLLOWED BY A PROLINE Chymotrypsin – carbonyl side of Phe or Tyr (aromatic) Elastase – carbonyl side of small, neutral residues Enzymatic Fragmentation 4 cut sites means 5 peptides The products of the reaction with trypsin are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the C-terminal end of the polypeptide. Substrate-binding Pocket Nature of pocket determines specificity Trypsin (basic) – negatively charged Chymotrypsin (aromatic) – hydrophobic (ser) Elastase (small) – bulky residues like Thr and Val at top and shallow Thr-226 Val-216 Asp-189 Substrate-binding Pocket Not active site Serine! Chymotrypsin Kinetics Assayed using an artificial substrate Useful as the nitrophenolate product absorbs at 400 nm Observed burst kinetics suggesting a first fast step and a slow second step Serine Proteases Display Burst Kinetics P1 (C-terminal region of peptide) Attack of water P2 (N- terminal region of peptide) Acyl-enzyme intermediate Serine Protease Mechanism A mixture of covalent and general acid-base catalysis Asp102 functions to orient His57 and forms a LBHB (Protonated His has a pKa similar to Asp) His57 acts as a general acid and base Ser195 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 the backbone N-H groups of Gly193 and Ser195 Substrate General base catalysis by binding His57 to form E-Ser195-S covalent intermediate Phe Collapse of unstable His57 stabilized by a LBHB tetrahedral intermediate and P release Stable inter. makes way Nucleophilic attack by water - for water His57 acts as a general base Collapse of unstable tetrahedral inter. and P Active site ready to repeat release (the sequel) Transition-State Stabilization The chymotrypsin mechanism involves two tetrahedral oxyanion transition states These transition states are stabilized by a pair of amide groups that is termed the “oxyanion hole” The Oxyanion Hole The O- (negative) in the tetrahedral oxyanion is stabilized by interaction with the backbone amide (NH) groups of Ser195 and Gly193 Catalytic Triads In General Found in several hydrolases and transferase enzymes Occur via divergent and convergent evolution Residues far away in primary structure Include: 1. Acid that orients and stabilizes the base (Asp, Glu, His) 2. Base that polarizes the nucleophile (His, rarely Lys) 3. Nucleophile that attacks substrate (Ser, Cys, sometimes Thr) Aspartic Proteases DIFFERENT structure and mechanism than serine proteases Active site contains two Asp (Pepsin res. 32 and 215) Cleave peptide bond between two hydrophobic amino acids No covalent catalysis Structures Structures of (a) HIV-1 protease, a homodimer, and (b) pepsin, a monomer. Pepsin’s N-terminal half is shown in red; the C-terminal half is shown in blue. Each lobe contributes one catalytic aspartate to the active site. pH-dependence pH profile Active at acidic pH Mechanism requires that one Asp be protonated and the other Asp be deprotonated when the substrate binds Importance of pKa A Mechanism for the Aspartic Proteases Tetrahedral intermediate Catalytic water LBHB allow “hydrogen tunneling” Mechanism for the aspartic proteases. LBHBs play a role in states E, ES, ET’, EQ’, and EP’Q. E.g. HIV-1 Protease A novel aspartic protease HIV-1 protease cleaves the polyprotein products of the HIV genome HIV-1 protease is a remarkable imitation of mammalian aspartic proteases HIV-1 protease is a homodimer - more genetically economical for the virus Active site is two-fold symmetric; different ‘flaps’ E.g. HIV-1 Protease Protease Inhibitors for AIDS Patients Protease inhibitors as AIDS drugs If HIV-1 protease can be selectively inhibited, then new HIV particles cannot form Structure-based drug design has developed several inhibitors that work in the dish Protease Inhibitors Block the Active Site of HIV-1 Protease HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck. The “flaps” that cover the active site are green; the catalytic active-site Asp residues are violet.