1. Enzymes are proteins that catalyze reactions.
2. Enzymes are capable of speeding reactions quadrillions of times faster than the same reactions would occur in the absence of enzymes.
3. Non-proteinaceous molecules that bind to enzymes and help the enzymes to catalyze reactions are called coenzymes.
4. The Gibbs free energy is the energy available to do useful work in reactions. The change in the Gibbs free energy for a reaction is important because it determines whether a reaction is favored (G <0), unfavored (G >0), or at equilibrium (G = 0).
5. Thus, when the Gibbs free energy change is negative, the reaction in question goes forward as written, but when the Gibbs free energy change is positive, the reaction goes in reverse.
6. A related term to G is G°', which is the Standard Gibbs Free Energy change. This refers to the Gibbs Free Energy change for a reaction under standard conditions. Since most reactions occur at non-standard conditions, G is much more useful than G°'. In fact, the sign of G°' does NOT tell the direction of a reaction, except under standard conditions.
7. Chemical reactions require activation energy (I'll call it G+ here) in order to get started. Catalysts (both enzymes and non-biological catalysts) act by lowering G+. Catalysts DO NOT CHANGE G. All they do is lower the energy required to activate the reaction. While enzymes speed reactions immensely, they therefore DO NOT CHANGE THE OVERALL REACTION CONCENTRATION AT EQUILIBRIUM. They simply allow the reaction to get to equilibrium faster.
8. G is affected by the concentration of reactants and products of a reaction by the following equation
G = G°' + RTln[Products/Reactants]
Thus, as product concentrations increase, the G will become more positive.
9. If one performs an experiment in which a fixed amount of enzyme is added to 20 different tubes, each containing a different amount of substrate (molecule that the enzyme catalyzes the reaction on) and then lets the reaction in each tube go for a fixed amount of time, one will create varying amounts of product when the tubes are analyzed. The greatest amount of product will be found in the tube which had the greatest amount of substrate. If one measures the concentrations of each product and divides by the time the reaction occurred, one obtains a velocity for each reaction. A plot of the velocity versus the substrate concentration (V versus S) from the experiment looks like the binding curve of myoglobin for oxygen - hyperbolic.
10. Velocity of an enzymatic reaction is measured as the concentration of product formed per time. Maximum velocity (Vmax) occurs in a reaction when the enzyme is saturated with substrate. Vmax depends on the amount of enzyme used to measure it.
11. In contrast to Vmax, Kcat is a constant for an enzyme. It is also known as the turnover number and corresponds to the number of molecules of product made per molecule of enzyme per second. 1000/second means 1000 molecules of product per molecule of enzyme per second. Kcat is calculated as Vmax/[Enzyme].
12. We define Km as the substrate concentration that gives Vmax/2. Whereas the Vmax varies, depending on the amount of enzyme that one uses, the Km is a constant for a given enzyme for its substrate.
13. The higher the Km of an enzyme, the LOWER its affinity for its substrate. This is because a high Km means that it takes a LOT of substrate before the enzyme gets to Vmax/2. Km is frequently referred to as the affinity of the enzyme for a substrate, though that is not 100% correct. Nevertheless, we say that a high Km is consistent with a low affinity of enzyme for substrate and conversely, that a low Km is consistent with a high affinity of enzyme for substrate.
14. If one lets a reaction go for a long time, it will reach equilibrium. At equilibrium, the relative concentration of products and reactants do not change. Initial velocities of reactions are therefore measured so as to avoid allow the product to accumulate and favor the reverse reaction.
15. The catalytic actions of enzymes appear to be related to their ability to be at least slightly flexible. Originally, Fischer proposed a model of catalysis called the Lock and Key model. It described enzymes as inflexible and the substrate as like a key fitting into a lock. While substrates do, in fact, fit into enzymes somewhat like a key, the enzyme is NOT inflexible.
16. Koshland's model of enzyme action, called the Induced Fit model says that not only does the enzyme change the substrate (via catalysis), but the substrate also changes the enzyme shape upon binding. This transient change of enzyme shape is important for catalysis because it may bring together molecular groups (such as a phosphate and a sugar) that may not be close together in the enzyme prior to the change in enzyme shape. Remember that enzymes also work by orienting substrates together in the proper way to maximize their likelihood of bouncing together in a way that leads to making a bond.
17. Chemical changes brought about by catalysis facilitate a last change in enzyme shape to allow for the release of the products. When this happens, the enzyme returns to its original shape and remains unchanged by acting to catalyze the reaction.
18. Most enzymatic reactions require binding of more than one substrate. I described two general mechanisms of catalysis - Sequential Displacement and Double Displacement.
19. Sequential displacement requires that substrates bind in a fixed order (Ordered Displacement) or in a random order (Random Displacement), but in either case, all of the substrates must bind before the process starts.
20. Double displacement reactions occur when one subrate binds, changes the enzyme in some way, dissociates, and then a second substrate binds and converts the enzyme back to its original state. The second substrate then dissociates.
21. Enzymatic reactions can be inhibited by reversible and irreversible processes. Reversible processes involve binding of an inhibitor and its subsequent release. Irreversible processes generally involve covalent attachment of a molecule to an enzyme followed by its inactivation. Irreversible inhibition can occur by mechanisms where the inhibitor has a structure similar to the substrate or where there is no relation between the two. In either case, the enzyme is destroyed for catalysis. The types of inhibition described below (competitive and non-competitive) are reversible.
22. Competitive inhibitors of enzymes are molecules whose structure resembles that of the normal substrate sufficiently that it is able to bind to the active site and stop the enzyme from functioning while it is bound there.
23. In competitive inhibition, the Vmax does not change because increasing amounts of substrate can swamp the inhibitor (present in fixed concentration), allowing the enzyme to effectively not see the inhibitor at high substrate concentrations. On the other hand, the apparent Km for competitive inhibition goes up because it takes more substrate to get the competitively inhibited reaction to Vmax/2.
24. On the other hand, non-competitive inhibitors do NOT bind to the active site of the enzyme and do not resemble the substrate. Therefore, addition of more substrate cannot eliminate the effect of the inhibitor. As a result, there is always a fixed amount of enzyme inactive in non-competitive inhibition. As you recall, when you change the amount of enzyme, you change the Vmax (from last lecture), so in the presence of a non-competitive inhibitor, the Vmax decreases.
25. In non-competitive inhibition, the Km does not change. This is because Km is a measure of the affinity of the enzyme for its substrate and this can only be measured by active enzyme. The fixed amount of inactive enzyme in non-competitive inhibition does not affect the Km and the Km, therefore is unchanged.