BL483 BASIC BIOCHEMICAL TECHNIQUES -- Lecture 3 - Enzyme Kinetics -- Expt 2 Part B
4. Simple Inhibitors of Enzymes and their Kinetic Analysis
Inhibitors of enzymes have a long history of usefulness in gaining understanding of how enzymes work and unraveling metabolic pathways. You will use two simple inhibitors in the lab this week: 1) the first is inorganic phosphate, Pi, which is the product of the phosphatase reaction and acts as a classical competitive inhibitor of the enzyme; and 2) fluoride, F-, which is an anion often found to inhibit phosphate metabolizing enzymes and acts as a classical noncompetitive inhibitor of the acid phosphatase. Well, what are competitive and non-competitive inhibitors of an enzyme. A competitive inhibitor is a substance which chemically resembles the substrate and its inhibition can be overcome by high concentrations of the substrate. A noncompetitive inhibitor is a substance with no chemical similarity to substrate and its inhibition can not be overcome by high concentration of substrate. Let us go back to our model of the kinetics of the phosphatase and see how these inhibitors act:
Competitive Inhibition:
Figure 5. Model for Competitive Inhibitor.
The competitive inhibitor, Ic, forms a complex with the enzyme at the substrate binding site and prevents the substrate from getting in. But the EIc complex is reversible and so as more substrate is added, the substrate manages to get in and overcomes the effects of the inhibitor. Consequently, the overall effect on the kinetics is to increase the Km and we call this new kinetic constant, the apparent Km or Km' (called Km prime or Km apparent). The maximum velocity which is attained is the same in the presence and absence of the competitive inhibitor, so Vmax is the same for both the uninhibited and competitively inhibited reaction. This is illustrated below using graphs for the response of an enzyme to competitive and noncompetitive inhibitors.
Non-Competitive Inhibition:
Figure 6. Model for Noncompetitive Inhibitor.
The noncompetitive inhibitor, Inc, forms a complex with enzyme, which is unaffected by the substrate concentration, such that it does not matter if substrate is already bound to the enzyme when the noncompetitive inhibitor binds or not. In this classical type of noncompetitive inhibition, the Km is not altered and only the Vmax is decreased. Thus, the real Vmax remains the same and you obtain an apparent Vmax or Vmax' (called Vmax prime or Vmax apparent).
Graph 3 and Graph 4 illustrate the expected results for a competitive and noncompetitive inhibitor.
Graph 3: v vs. [S] plot for competitive and noncompetitive inhibitors.
Graph 4: Double Reciprocal plot for competitive and noncompetitive inhibitors.