Amp Synthesis Lab

Abstract This experiment investigated the kinetics of the enzyme glycogen phosphorylase b which is important to metabolism. AMP is an allosteric activator of the enzyme because it converts glycogen phosphorylase b from its T state to the R state which is the active form. Caffeine is an inhibitor because it binds the nucleoside inhibitor site. When it binds this site, it stabilizes the inactive T state and blocks the catalytic site which needs to be open for enzyme activity to occur. The glycogen phosphorylase b was purified with hydrophobic column chromatography and the concentration was determined with a Bradford Assay. The kinetics of glycogen phosphorylase b were studied by finding a molar extinction coefficient, 0.1617 mM cm-1 A-1, for
These regulators effect the activity of the phosphorylase but not the actual reaction. Regulators bind allosteric sites in order to effect the enzyme activity usually through conformation changes. The activity can either be increased or decreased depending on whether the regulator is an effector or inhibitor for the enzyme. An important allosteric effector for glycogen phosphorylase is AMP. AMP will bind an allosteric site with high affinity to convert the glycogen phosphorylase from a T (inactive) form to the R (active) form of the enzyme. Once the enzyme is activated by AMP it can assist in the degradation of glycogen. Glycogen phosphorylase can also be inhibited by regulators. One inhibitor of the enzyme is caffeine. Caffeine is a related heterocyclic ring compound that will bind the nucleoside inhibitor site of the enzyme. It has a dual action of stabilizing the inactive T form of the enzyme and blocking the catalytic site needed for activation. Caffeine works in concert with glucose so it will promote the binding of glucose and vice versa. Caffeine and glucose act as inhibitors for both glycogen phosphorylase a and b
Kinetic constants are a good way to compare the effects of regulators on the enzyme. These kinetic constants include Vmax and Km. Vmax represents the maximal reaction rate and is the plateau of a Michaelis-Menten curve. At this point, the active sites are saturated or no longer as accessible due to conformational change. The Km is the Michaelis constant and is an inverse of the substrate’s affinity for the binding site. As an inverse, a lower Km represents a higher substrate affinity for the enzymatic binding site. Inhibitors should result in a lower Vmax due to their effects on the activity of the enzyme, while effectors should increase the enzymatic