Highlights Lecture #8 Spring 2017

1. 2D gel electrophoresis involves two separations of proteins in two dimensions. Each dimension employs a different separation strategy. The first involves separation on the basis of charge in a technique called isoelectric focusing. It is performed in a column/tube. The second technique employs SDS-PAGE. It involves placing the tube containing the separated charged molecules across the top of an acrylamide gel, adding SDS, and then running the contents on SDS-page. The resulting gel has molecules separated by size in the vertical dimension and by charge in the horizontal dimension.

2. HPLC (High Performance Liquid Chromatography) is another technique for separating molecules/proteins. It works by separating on the basis of polarity using specialized columns that perform reversed phase separations. In reversed phase separations, molecules that are the least polar elute from the column last, whereas molecules that are the most polar elute first.

3. Proteins can be broken/digested into smaller pieces using enzymes like trypsin (cuts lysine or arginine) or chymotrypsin. They can also be broken by chemical agents, such as 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 – 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 can hold them for a reaction. An “active site” of an enzyme is a site on an enzyme where the reaction it catalyzes occurs.

5. As one increases the amount of substrate for an enzymatic reaction, the velocity of the reaction (concentration of product made per time) increases. If one uses more enzyme, one produces a faster velocity.

6. 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.

7. Vmax varies with the amount of enzyme used in a reaction. To account for the amount of enzyme in a reaction, Kcat (also called turnover number) is calculated. Kcat is equal to Vmax/[Enzyme]. Because the concentration of enzyme is taken into account in this equation, Kcat does NOT vary with the amount of enzyme used and is therefore a constant for an enzyme. Kcat is equal to the number of molecules of product made per enzyme per unit time. A Kcat of 5/second means that that enzyme makes five molecules of product per molecule of enzyme per second.

8. A very important number that does NOT vary according to the quantity of enzyme used (that is to say that it is a constant for a given 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 is a constant for any given enzyme and 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.

Print Friendly, PDF & Email