Highlights Enzymes II
1. The active site of an enzyme is the place on the enzyme where the reaction is catalyzed. The substrate binding site is immediately adjacent to it.
2. Temperature affects an enzymatic reaction in two ways. First, temperature is a factor in the Gibbs Free Energy, which determines the energy of a reaction. Second, temperature can denature an enzyme and thus negatively impact an enzyme’s activity.
3. There are two models for the mechanism of enzyme action relevant for our consideration. The “lock and key” model proposes that enzymes act like a “lock” that only certain keys (substrates) fit. This model works well for describing the binding of substrates, but is not helpful (or accurate) for describing the mechanism of an enzyme’s catalytic action.
4. The “induced fit” model of enzyme action proposes that enzymes change in response to binding of substrate and that change is at least partly responsible for the catalysis that occurs on the substrate. Thus, the induced fit model says that enzymes change substrates (by catalysis) and that substrates also change enzymes (enabling catalysis). The lock and key mode, by contrast,l says that an enzyme changes a substrate, but says nothing about how it does it. On the other hand, the Koshland Induced Fit says that not only does an enzyme change a substrate, but that a substrate also transiently changes an enzyme.
5. The “tension” that an enzyme experiences during its binding of a substrate helps to cause the substrate to be modified (reacted). It is important to note that after catalysis occurs, the product is released and the enzyme is returned to its original state.
6. Some enzymes have their ability to catalyze a reaction affected by the presence of another molecule. If that molecule is the substrate, one obtains a sigmoidal plot for V vs [S] like that of hemoglobin binding to oxygen. This type of plot is is evidence that the enzyme’s activity is affected by the substrate. When the activity of an enzyme is affected by binding a small molecule, the enzyme is described as allosteric. Allosterism specifically means that binding of a small molecule to an enzyme affects the enzyme’s activity.
7. A very important number for an enzyme is the Km (the Michaelis constant). Km turns out to be the concentration of substrate required to get an enzymatic reaction to half maximum velocity. Km provides a measure of an enzyme’s “affinity” for its substrate. An enzyme with a high Km has a low affinity for its substrate. An enzyme with a low Km has a high affinity for its substrate. Note that Km is NOT Vmax/2. Instead, it is the substrate concentration required to get a reaction to Vmax/2.
8. A “perfect enzyme” is one that has a maximum value for Kcat/Km. These enzymes have maximaly velocities and minimal Km values (high affinity). Perfect enzymes are slowed only by the rate with which the substrate can diffuse to the active site of the enzyme.
9. Determining Vmax from a plot of V versus S is not easy. Consequently, an alteration of this plot is done to make the measurement simpler. The most common alteration is known as a Lineweaver-Burk (double-reciprocal) plot. In it, a double reciprocal plot is performed – 1/V versus 1/S. When this is plotted for an enzymatic reaction, a line is produced, with the x-intercept (place where the line intersects the x-axis) equaling -1/Km and the y-intercept (place where the line intersects the y-axis) equaling 1/Vmax.
10. Inhibition of enzymes occurs competitively when the inhibitor of the enzyme resembles the substrate and competes with the substrate for binding to the enzyme. Because they are competing with each other for the same site on the enzyme, the substrate can “overcome” the inhibitor at high concentrations (because reactons are set up with a fixed amount of inhibitor). Thus, although it will require more substrate in the presence of an inhibitor to get the same enzyme reaction velocity as when there is no inhibitor, the Vmax of a competitive inhibition is unchanged from the uninhibited reaction.
11. Note above that “it will require more substrate in the presence of an inhibitor to get the same enzyme reaction velocity as when there is no inhibitor” so the Km will change. It requires MORE substrate to get the reaction with the inhibitor to half maximum velocity (compared to uninhibited). Therefore, the Km for a reaction undergoing competitive inhibition INCREASES, meaning the affinity of the enzyme for substrate DECREASES.
11. In non-competitive inhibition, the inhibitor binds to a site on the enzyme that is NOT related to the substrate. Therefore, substrate and inhibitor are NOT competing for the same site on the enzyme. This means then that increasing the substrate concentration does NOT affect the inhibitor’s ability to inhibit the enzyme. It also means that a fixed percentage of the enzyme is always inactivated by a non-competitive inhibitor. In non-competitive inhibition, the apparent Km is the same as the uninhibited reaction, whereas the Vmax decreases.
End of Material for Exam 1