Biochemistry I Lecture 18 PDF

BIOL or CHEM 3361 Biochemistry I Mechanisms of Enzyme Action Reading: Chapter 14 Mechanisms of Catalysis 1) Catalysis by proximity (or formation of near-attack complexes) 2) Covalent catalysis 3) General acid-base catalysis 4) Metal ion catalysis Also, low barrier hydrogen bon...

BIOL or CHEM 3361 Biochemistry I Mechanisms of Enzyme Action Reading: Chapter 14 Mechanisms of Catalysis 1) Catalysis by proximity (or formation of near-attack complexes) 2) Covalent catalysis 3) General acid-base catalysis 4) Metal ion catalysis Also, low barrier hydrogen bonds 1) Catalysis by Proximity Enzymes bring substrates in the active site close enough and in the proper orientation such that their collision will occur at a higher frequency Absence of enzyme: 0.0001% Presence of enzyme: 1 to 70% Active site is “pre-organized” to form near-attack complexes where reacting atoms are in van der Waals contact at an angle resembling the bond to be formed in the transition state Near-attack Complexes NACs are characterized as having reacting atoms within 3.2 Å and an approach angle of ±15° of the bonding angle in the transition state. Alcohol dehydrogenase – a near-attack complex. Protein Motions Are Essential to Enzyme Catalysis Proteins are constantly moving – bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit Active site conformation changes can: Assist substrate binding Bring catalytic groups into position Induce formation of NACs Assist in bond making and bond breaking Facilitate conversion of substrate to product 2) Covalent Catalysis Active site residues form a temporary covalent bond with the substrate. At the end of the reaction the covalent bond is broken to regenerate the enzyme. Often occurs due to nucleophilic attack by amino acid side chains on electrophilic groups on substrates Can also involve prosthetic groups Facilitates electron transfer Most have an unknown mechanism Double Displacement Double displacement: the two substrates bind and react separately in a ping pong manner E’ is a covalently modified enzyme intermediate! Reactions conforming to this kinetic pattern are characterized by the fact that the product of the enzyme’s reaction with A (called P in the above scheme) is released prior to reaction of the enzyme with the second substrate, B. E.g. Enzyme-substrate Intermediates 2) Covalent Catalysis 3) General Acid-Base Catalysis Is the transfer of a proton in the transition state. Transferring an H+ may: 1. Activate nucleophiles 2. Stabilize charged groups 3. Improve electrostatic interactions that stabilize TS Facilitates proton transfer Specific would means H+ or OH- that has diffused into the active site Candidate amino acids are Glu, Asp or His Histidine Often Plays a Role An active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate. Water Often Plays a Role Water can act as an acid or base at the active site through proton transfer with an assisting active-site residue Shifts in pKa for Residues in Active Sites Create Catalytic Agents From Creighton, Proteins (1993) Primary: His, Cys, Asp, Glu, Arg, Lys, Tyr, Ser, Thr, Asn, Gln Secondary Roles About half of the amino acids engage directly in catalytic effects in enzyme active sites Other residues may function in secondary roles in the active site: Raising or lowering catalytic residue pKa values Orientation of catalytic residues Charge stabilization Proton transfers via hydrogen tunneling 4) Metal Ion Catalysis e.g. zinc, magnesium, iron Metal atoms lose electrons easily and exist as cations Positive charge allows them to: - stabilize transient and intermediate structures - assist in forming strong nucleophilic species (image) - hold substrate inside the active site - stabilize charge Also, Low-Barrier Hydrogen Bonds (LBHBs) The typical H-bond strength is 10-30 kJ/mol, and the O-O separation is typically 0.28 nm As distance between heteroatoms becomes smaller (