BCH 219L Enzyme Assay Background PDF

BCH 219L Enzyme Assay Background Adapted by Amanda Vines from ACC BIOL1415 lab manual by J. O’Grady et al. ENZYMES One of the more essential functions of proteins is that of catalysis. Enzymes are biological molecules that help reactions in the cell to occur up to 1020 times faster tha...

BCH 219L Enzyme Assay Background Adapted by Amanda Vines from ACC BIOL1415 lab manual by J. O’Grady et al. ENZYMES One of the more essential functions of proteins is that of catalysis. Enzymes are biological molecules that help reactions in the cell to occur up to 1020 times faster than they normally would on their own! Without the aid of enzymes, these reactions would take place too slowly for the organism to survive! Except for catalytic RNA, most enzymes are globular proteins. Enzymes mediate virtually all chemical reactions of living organisms. Therefore, their characterization is crucial for the understanding of biology. The three-dimensional structure of the enzymes creates a characteristic pocket or active site into which the substrate or substrates fit. This allows for the reaction to occur without using up the enzyme—hence it is a catalyst. The rate at which an enzyme catalyzes a reaction is characteristic for that enzyme at a given pH, temperature and ionic strength. Therefore, rate kinetic studies are often performed on enzymes. In this lab, students will perform kinetic studies of the enzyme Alkaline Phosphatase. Phosphatase is an enzyme that removes a phosphate group from a molecule. The substrate used in this experiment is p-nitrophenyl phosphate. The velocity of the reaction is monitored by measuring the absorbance of the colored product at 405 nm in a spectrophotometer. Km and Vmax The value of Knowing Km and Vmax for an Enzyme: Understanding how an enzyme works and how an inhibitor prevents an enzyme’s function are important, especially in the pharmaceutical industry. Two important terms to know for an enzyme are its Km and Vmax. But what can Km and Vmax tell you about an enzyme? Km is the substrate concentration that is required for the reaction to occur at half Vmax. A high Km means it takes a greater concentration of substrate to be half saturated. For most enzymes, Km lies between 10-1 and 10-7 M and depends on the substrate and environmental conditions such as pH, temperature, and ionic strength. Km is also related to the rate constants of the individual steps in the catalytic scheme (i.e., the formation and dissociation of the enzyme-substrate complex). See the generalized reaction below, where E = enzyme, S = substrate, and P = product. E + S  ES  E + P Under conditions where the dissociation of ES is much greater than the formation of P, Km is a measure of the strength of the ES complex. If Km is high, binding affinity is small. The maximal velocity of the enzyme, Vmax, reveals the turnover number of an enzyme, which is the number of substrate molecules converted into a product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate. The turnover numbers of most enzymes with their physiological substrates fall in the range of 1 to 104 per second. A low Vmax means the enzyme does not convert as much substrate to product. 1 INHIBITORS Though we won’t use inhibitors in our enzyme assay in lab, it’s important to know about how they can affect enzyme kinetics. The two main types are competitive inhibitors and noncompetitive inhibitors. Using Lineweaver-Burk plots, one can distinguish between inhibitors. A typical Lineweaver-Burk plot of an enzymatic reaction is given below. Remember from your chemistry courses that a Lineweaver Burk plot is a reciprocal plot of the Michaelis Menten plot (y axis: V and x axis: [S]) and allows for better quantification of Km and Vmax with enzymatic data. Figure 1. An annotated general Lineweaver-Burk plot. Retrieved from https://www.chem.purdue.edu/courses/chm333/Spring%202013/Lectures/Spring%202013%20Lecture%2015.pdf Competitive inhibitors are molecules that sit in the active site of the enzyme and prevent entry of the substrate. Therefore, competitive inhibitors frequently have a similar structure to the substrate. A Lineweaver-Burk double reciprocal plot of a competitive inhibitor will have the same Y-intercept as that of the substrate. Vmax remains the same; it is unaffected with a competitive inhibitor because higher substrate concentrations can overcome the presence of the inhibitor. Km increases with a competitive inhibitor because it is competing with the active site. Figure 2. A generalized Lineweaver-Burk plot comparing slopes of enzyme reactions with no inhibitor and one portion (+I) or two portions (+2[I]) of a competitive inhibitor. Note that the lines converge on the y intercept since Vmax, and therefore 1/Vmax, is unchanged. 2 Noncompetitive inhibitors do not sit in the active site but will attach to an allosteric site and change the shape of the enzyme and therefore alter the active site. The changed enzyme shape results in preventing the substrate from being converted into the product when bound to the enzyme. Km (binding affinity) is unaffected since the inhibitor does not affect binding to the active site. Vmax decreases with a non-competitive inhibitor because it does not compete with the active site. In a Lineweaver-Burk double reciprocal plot of a noncompetitive inhibitor, the X-intercept, -1/Km is the same as that for the substrate. Thus, the two types of inhibitors may be distinguished from each other. Figure 3. A generalized Lineweaver-Burk plot comparing slopes of enzyme reactions with no inhibitor and one portion (+I) or two portions (+2[I]) of a noncompetitive inhibitor. Note that the lines converge on the x intercept since Km, and therefore -1/Km, is unchanged. 3