1. HPLC (High Performance Liquid Chromatography) is another technique for separating molecules/proteins. It works by separating on the basis of polarity. Molecules that are the least polar elute from the column last, whereas molecules that are the most polar elute first.
2. Proteins can be broken into smaller pieces using enzymes like trypsin (cuts lysine or arginine), chymotrypsin, and cyanogen bromide (breaks at methionines).
Highlights Enzymes I
1. Enzymes catalyze reactions up to many trillion of times faster than the same reactions without any catalysts.
2. Enzymes work by reducing the activation energy required for a reaction to occur. Because enzymes lower that "starting" energy requirement, they make the reaction easier to occur and thus speed them up.
3. Note also (important) that enzymes do NOT change the free energy difference between the beginning reactants and the end products. Thus, enzymes do not change the overall energy of a reaction or the equilibrium of a reaction - only the energy required for the transition state.
4. A "substrate" is a molecule bound by an enzyme which it catalyzes a reaction upon. Substrates bind specific binding sites on enzymes that resemble their structure. An "active site" of an enzyme is a site on an enzyme where the reaction it catalyzes occurs.
5. 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 catalysis.
6. 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 change enzymes (enabling catalysis). Thus, the lock and key model 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.
7. 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.
8. As one increases the amount of substrate for an enzymatic reaction, the velocity of the reaction (concentratioin of product made per time) increases. If one uses more enzyme, one produces a faster velocity.
9. An enzymatic reaction's maximum velocity (Vmax) is the limit (maximum) of a plot of the velocity versus the substrate concentration. Enzymatic reactions reach maximum velocity when the enzyme is saturated with substrate. Plots of enzyme velocities versus substrate concentration are called hyperbolic.
10. Vmax varies with the amount of enzyme used in a reaction.