C430 Enzymes (intro & kinetics)

• 1. Biological catalysts that increase the rate of rxns (w/o affecting equilibrium) but are unchanged at the end of the rxn.
• 2. Exergonic
• 3. increase in rxn rate, increase in frequency of collisions of reactive molecules, allows rxns to occur under mild conditions (not heated, etc), and regulates rxns (on/off)

• 1. 10^3 to 10^17 faster
• 2. By acting as a platform for reactive molecules to interact with correct orientation and proximity.
• 3. Usually, to increase rxn rate, we have to increase temp and pressure. But this way, with enzymes, can increase rxn rate at normal conditions for humans (pH = 7, 37 C, 1 atm).

1. Transient (only present during rxn) or integrated into 3D structure (prosthetic group)

• 1. Oxidoreductases - redox
• 2. Transferases - transfer of functional group
• 3. Hydrolases - hydrolysis (break bond with H2O)
• 4. Lyases - add or remove groups to make or break C=C.
• 5. Isomerases - rearrange C skeleton
• 6. Ligases - form C-C, C-N, C-O, and C-S bonds with ATP.

• 1. Can change k (rate constant), can't change Keq.
• 2. Because
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• and can't change Keq without changing dG, which would break the law of thermodynamics.
• 3. 1 atm, 25 C, 1 M of reactants (for biochemists, pH = 7 too).
• 4. As Keq increases, dG becomes more negative
• 5. As the rxn is more product-favored, the rxn becomes more energetically favorable (exergonic and spontaneous)

• 1. dG deals with energy (exer/endergonic) while dH deals with heat. (exo/endothermic)
• 2. yes, because it just comes down to dG.
• 3. (1) entropy - reduces possibility that they'll react with each other (2) solvation shell of H-bonded H2O that stabilizes most biomolecules (3) distortion of substrates

1. Binding energy dGB

2. (1) Rearrangement of covalent bonds between enzymes & substrates and (2) Formation of weak, noncovalent interactions between substrate and enzyme.

• 3. Specificity and catalysis
• 4. As surface area of contact between enzyme and substrate increases, binding energy increases too.

• 2. To keep interacting groups properly positioned to favor transition state
• 3. Keep cavity from collapsing.

• 1. Increases entropy by displacing solvent molecules from binding pocket.
• 2. Summation of unfavorable (positive) activation energy and favorable (negative) binding energy results in lower net activation energy.
• 3. Weak binding interactions that are formed between substrate and enzyme.

• 1. Proximity and orientation
• 2. Preferential transition state binding
• 3. Acid-base catalysis
• 4. Covalent catalysis
• 5. Electrostatic catalysis
• 6. Metal ion catalysis

1. Enzymes bind so that reactive groups are close and in correct orientation by constraining motions of substrates (reducing entropy) increasing possibility of rxn.

2. Enzymes preferentially stabilize transition state over substrate by inducing conformational change in enzyme "induced fit" model.

• 1. ES will be at lower energy level than product (b/c release of energy due to formation of bonds to stabilize). Basically, would require more energy to go from ES --> P than S--> P, defeating purpose of using enzyme.
• 
• 3. Would have to overcome original dGcat with dGM on top.

• 1. Complex has enough energy to overcome activation barrier, preventing reverse rxn from occurring. (Enzyme increases number of substrates in forward rxn to prevent reverse rxn)
• 2. dGM is the difference between the transition-state energies of the uncatalyzed and catalyzed rxns.

• 1. Acid-base: side chains function as proton donors and acceptors.
• 2. Transient (eventually must be broken) covalent bond forms between enzyme and substrate
• 3. Certain R groups stabilize high energy intermediates
• 4. Seen in redox rxns, involved in substrate binding.

• 1. Asp 1 and 2 - stabilize His 57 (proximity and orientation)
• 2. his 57
• 3. Ser 195

His and Ser are responsible for actual chemistry steps in catalysis.

• 2. Covalent catalysis (may also have electrostatic for subsequent stabilization).
• 3. They often look very similar, but acid-base catalysis would be the actual transfer of a proton, while electrostatic catalysis is where the proton just surrounds the negatively charged anion.
• 4. Stabilizes TS preferentially

• 1. No, need to know intermediates.
• 2. Rate equation, i.e., rate = k[A][B]
• 3. B/c requires simultaneous collision of three substrates.
• 4.

• 1. none - all enzymatic rxns can also go in reverse.
• 2. At first, increase in substrate leads to linear & proportional increase in product, but at a certain point, all enzymes are saturated (assuming constant [E]) and it plateaus. This plateau is Vmax.
• 3. Beginning = first order rxn. End = zero order rxn (no matter how much more substrate you add, it doesn't matter).

• 1. Assumption of equilibrium - measurements are taken early in rxn, so we can assume that K-2 doesn't play a role (no equilibrium).
• 2. Steady state - the initial part of rxn reflects steady state in which [ES] remains constant; formation of ES must equal breakdown of ES.
• 2. No
• 3.
• 4. ES --> Product; depends on [ES].

Rate = k2[ES]

• 1. E + S <--> ES ---> E+P (k1, k-1, k2)
• 2.
• 3.
• 4.
• 5. Because it simplifies equation.

• 1.
• 2.
• 3. Km represents enzyme's affinity for substrate (like Kd for proteins).
• 4. Km is unique for every pair.
• 5. High Km represents less affinity, low represents high affinity.

• 1. Because it's very difficult to pinpoint, especially on an asymptote.
• 2. Linearize data into double reciprocal plot to get more accurate numbers.
• 3.
• 4.
• 5. We assume [S]>>[E]

• 1. Vmax stays constant while Km increases (less affinity)
• 2. Vmax decreases while Km stays constant.

For competitive inhibitor (CI), assuming same binding affinity for substrate, Km increases b/c it will require more S to reach Km point (1/2 of Vmax) Vmax stays constant b/c competitive inhibition can be overcome by sufficiently high [S]

For noncompetitive inhibitor - Vmax decreases as a result of removing activated complex, but since I doesn't interfere with binding site, Km stays the same. It doesn't matter how much substrate is added, Vmax will decrease.

• 1. Distinguishing between certain types of enzymatic rxns and analyzing enzyme inhibition.
• 2. Hb binding affinity will increase, b/c BPG is negatively charged and would not be able to bind as well to alanine. Curve will look more like myoglobin.
• 3. B/c of remaining salt bridges
• 4. By mutating certain ones and seeing hte result.

• 1. When [S]=Km
• 2. Shifts it to the left. Shifts it to the right.