This week’s homework assignment is now posted.

Web-based simulations and animations are also on the Week 6 page.

Laboratory • Lab this week involves an experiment with enzymes that you and your lab partners will design — please read the lab manual ahead of time to prepare!

Exams and Labs • Requests for regrades may be left in the box just past my office (Sidney Frank Hall 211). • All regrade requests filed to date are finished, and may be picked up, right next to the drop-off point. Unclaimed exams are also outside SFH 211, right here:

Let’s fight cancer together! relayforlife.org/brown Relay for Life Register this week! Let’s fight cancer together! Entertainment all night, food all night! Relay for Life! April 12th at the OMAC! 6am-6pm relayforlife.org/brown

Enzymes Basic principles of catalysis substrate binding catalytic mechanisms Inhibition competitive non-competitive Feedback inhibition

∆G° = -4.0 kCal/mole (slight release of energy) Problem: many chemical reactions are slow. NH2 H2O 2 (NH3) CO2 + C = O + reactants products urea + water carbon dioxide + ammonia ∆G° = -4.0 kCal/mole (slight release of energy) 98°C, 2-3 hours to reach equilibrium in test tube Solution: Add an “enzyme:” Urease. 37°C, equilibrium is reached in 30 seconds. What, exactly is an “enzyme,” and how do they speed up reactions?

If products have a lower energy level, why doesn’t the reaction occur spontaneously? Urea Ammonia Because the reactants first have to be raised in energy to reach a transition state. Energy to do this is known as “activation energy.”

Catalysts are compounds that lower activation energy: Lowering Ea lowers the temperature needed to reach Ea and speeds the rate at which it reaches equilibrium. But: it does not change the energy levels of product or reactant.

Enzymes are Biological Catalysts Basic Principles of Enzyme Catalysis: Enzymes lower the activation energy of reactions Enzymes do not change the overall energetics of a reaction Enzymes bind their substrates (reactants) at an “active site” Enzymes catalyze reactions by chemical interaction with their substrates Enzymes are usually proteins (Although RNA can also act as a catalyst)

Adenosine Triphosphate (ATP) • Enzymes do not change the overall energetics of a reaction. So how do cells carry out energy-requiring (endergonic) reactions? Adenosine Triphosphate (ATP)

Substrate (reactant) is converted into product while tightly bound to the enzyme E + S ES EP E + P

Lysozyme (catalyzes the cleavage of carbohydrates in cell walls of bacteria) Active Site The substrate binds tightly & specifically to the active site

Enzyme catalyze reactions by Stabilizing the Transition State in several ways: 1) Proximity effect (bringing substrates close to each other) 2) Orientation (binding substrates in proper orientation for reaction) 3) Strain (binding imposes forces that strain or break existing bonds) 4) Direct chemical interaction (direct transfer of ions or electrons to substrates)

Example #1 : a cyclic AMP protein kinase. It catalyzes transfer of a phosphate group from ATP to another protein “target” peptide ATP

Example #1 : a cyclic AMP protein kinase • Both substrates bind to active site (Lysine & Mg++ pull at phosphate group electrons) • New bond forms between serine of substrate & third phosphate group • These interactions break bond between 2nd and 3rd phosphates, transferring the 3rd phosphate to serine on substrate peptide

Example #2: The action of lysozyme, which breaks a glycosidic bond between two sugars in bacterial cell wall polysaccharides. Sugar “D” is forced into a strained configuration. Glu- 35 donates a H+ ion, breaking the glycosidic bond, and a water molecule completes the cleavage reaction.

Example #3: The enzyme Ribonuclease binds RNA and breaks the sugar- phosphate bond between two nucleotides: from: Freeman 3/e p. 61

Enzymes can be active participants in chemical reactions Enzymes can be active participants in chemical reactions. Example: Hexokinase binding its substrate: “Induced fit:” substrate binding to active site causes a conformational change (change in shape) making the binding tighter.

“Induced fit” as Hexokinase binds substrate.

Example: ethylene glycol. Enzyme converts it into oxalic acid (toxic) Enzyme inhibition: A competitive inhibitor competes for binding to activate site. (it competes with substrate). — OH Example: ethylene glycol. Enzyme converts it into oxalic acid (toxic) CH2 Substrate Inhibitor COOH

CH2 — OH CH2 — H — OH ethanol

HIV produces a single large “polyprotein” that must be cut into smaller, functional polypeptides. Block the enzyme that does this (HIV-protease), and you have a way to stop the virus. Strategy: Design a molecule that fits into the active site of the enzyme - a competitive inhibitor.

How effective has the inhibitor approach been to HIV therapy?

Science 27 August 2004: Vol. 305. pp. 1243 - 1244 Combined HIV therapy, including protease inhibitors and retrovirals, has dramatically reduced AIDS deaths. Science 27 August 2004: Vol. 305. pp. 1243 - 1244

Another form of inhibition: A non-competitive inhibitor binds to an enzyme somewhere other than the activate site. (it does not compete with substrate). It acts (usually) by causing a conformational change in the enzyme that alters the active site. Non-competitive inhibitors are just one example of molecules that influence enzyme activity by allosteric effects (by changing the molecule’s conformation)

Non-competitive inhibitors are just one example of molecules that influence enzyme activity by allosteric effects (by changing the molecule’s conformation)

From your textbook: Allosteric Regulation Competitive Inhibition Non-Competitive Inhibition Allopsteric Regulation can be positive (Activation) or negative (Deactivation or Inhibition)

Example: Isoleucine inhibition of the enzyme threonine deaminase. Many biochemical pathways are also regulated by allosteric (non-competitive) inhibition. When the end- product of a pathway is the inhibitor, it produces an effect known as “feedback inhibition.” Example: Isoleucine inhibition of the enzyme threonine deaminase.

Why does this make sense?? Feedback Inhibition frequently is the result of end-product binding to an allosteric site. The end-product acts as a non-competitive inhibitor Question: Why does this make sense??

Metabolism - the sum total of chemical pathways in the cell