Reaction Mechanisms

1. Reaction Mechanisms C, D, E, H, S WHY? • The catalytically important amino acids are? • In the protease mechanisms we have reviewed, the carbonyl carbon on the peptide bond is the target. • If you are given the catalytic amino acids of a protease, remember the target and remember the products of the protease reaction: 2 peptides • In the lysozyme mechanism, the reaction is started by protonation of the glycosidic oxygen • We need to have products that have the hydroxyl groups attached to them as we know carbohydrates should (polyhydroxylaldehydes or ketones)

2. Chymotrypsin Mechanism

3. Different Active Site, Slightly Different Mechanisms Chymotrypsin is a protease, specifically a Serine Protease There are other types of proteases: • Cysteine Proteases • Cys residue replaces Ser in mechanism similar to Serine proteases • Aspartic Proteases • 2 Asp residues act as General Acid-base catalysts • Zinc Proteases • Zn2+ is coordinated by 2 His • Zn2+ promotes attack of carbonyl carbon by water

4. Alcohol DehydrogenaseMechanism Steps • Binding of the coenzyme NAD+ • Binding of the alcohol substrate by coordination to zinc • Deprotonation of nicotinamide ribose by His-51 • Deprotonation of Ser-48 by nicotinamide ribose • Deprotonation of the alcohol by Ser-48 • Hydride transfer from the alkoxide ion to NAD+, leading to NADH and a zinc bound aldehyde or ketone • Release of the product aldehyde

5. Alcohol DehydrogenaseMechanism Start at bottom and work your way clockwise, Follow the electrons!

6. Alcohol DehydrogenaseQuestions for Your Consideration • How effective do you think the enzyme will be with various alcohols as substrate? • What effect do you think performing the reaction at an acidic pH would have? Basic pH? • If you mutated Ser48 to a Threonine, what would happen to the observed activity? • Turn your answers in next Tuesday (March 9).

7. Membrane Function: Membrane Transport Passive transport • driven by a concentration gradient • simple diffusion: a molecule or ion moves through an opening • facilitated diffusion: a molecule or ion is carried across a membrane by a carrier/channel protein • Active transport • a substance is moved AGAINST a concentration gradient • primary active transport: transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ ion pump • secondary active transport: driven by H+ gradient

8. Passive Transport • Passive diffusion of species (uncharged) across membrane dependent on concentration and the presence of carrier protein

9. 1˚ Active transport • Movement of molecules against a gradient directly linked to hydrolysis of high-energy yielding molecule (e.g. ATP)

10. Membrane Receptors • Membrane receptors • generally oligomeric proteins • binding of a biologically active substance to a receptor initiates an action within the cell

11. Oxidation Reactions • Involves the transfer of electrons (OIL RIG): • oxidation being termed for the removal of electrons • reduction for gain of electrons Loss of electrons or hydrogen = oxidation Gain of electrons or hydrogen = reduction • Oxidation is always accompanied by reduction of an e- acceptor • Cells (plants and animals) rely on O2 for life processes • Water an electron acceptor in plants • Animal cells generate water from the reduction of O2 by H+

12. Oxidation Reduction Reactions Fe 2+ + Cu 2+  Fe 3+ + Cu + Reaction can be expressed in the form of 2 half reactions Fe 2+  Fe 3+ + e- (oxidized); Fe 2+ = reducing agent Cu 2+ + e- Cu + (reduced) ; Cu 2+ = oxidizing agent Reducing agent = e- donating molecule Oxidizing agent = e- accepting molecule They together make a conjugate redox pair.

13. Redox Potential • Also known as oxidation reduction potential • Redox potential of any substance is a measure of its affinity for electrons • In oxidation/reduction reactions the free energy change is proportional to the tendency of reactants to donate / accept e- denoted by E°’ ( for biological systems) • A reaction with a positive E°’ has a negative Go’ (exergonic) • The redox potential of a biological system is usually compared with the potential of Hydrogen electrode expressed at pH 7.0

14. Reduction potentials • A reduction potential is a measure of the affinity of an atom for electrons • Electrons are a standard currency that let us rank the reducing/oxidizing potential of different redox couples. • When the difference between the E°’ values is positive, then G° is negative because G°=-nFE°’ • The more positive the standard reduction potential E°’, the greater the tendency for the redox couple’s oxidized form to accept electrons and become reduced. • Electrons flow towards the half cell with the more positive E°’

15. Reduction of NAD+ by FADH2 Consider the following reaction: NAD+ + FADH2 --> FAD + NADH + H+ 1st Half Reaction: NAD+ + H+ + 2e- --> NADH E°’ = -0.320V 2nd Half Reaction (Note: Its reversed!): FADH2 -->FAD + 2H+ + 2e- E°’ = +0.219V E°’= –0.320V + +0.219V = -0.101V. Since E is negative, G is positive and the reaction is not spontaneous. Thus, FADH2 cannot be used to reduce NAD+.

16. Reduction of FADbyNADH Consider the following reaction: NADH + H+ + FAD --> FADH2 + NAD+ 1st Half Reaction (Note: Its reversed!): NADH --> NAD+ + H+ + 2e- E°’ = +0.320V 2nd Half Reaction: FAD + 2H+ + 2e- --> FADH2 E°’ = -0.219V E°’= +0.320V + -0.219V = +0.101V. Since E is positive, G is negative and the reaction is spontaneous. Thus, NAD+ can be used to reduce FADH2.