Question

4. The rate with which enzymes can transform a substrate varies from enzyme to enzyme, and sometimes substrate to substrate. a) How are competitive and uncompetitive reversible inhibition similar? How is their effect on rate different? b) Enzyme reaction rates drop off dramatically as temperatures pass 50 °C or so, and do not return to their previous rate when cooled. Explain the drop off in activity and the lack of return to the previously higher reaction rates after heating. c) Pepsin is an enzyme that participates in digestion in the stomach, whereas trypsin is an enzyme that participates in digestion in the small inestines. Pepsin has the highest turnover number at around pH 2, whereas trypsin has the highest turnover number around pH 8. Explain this difference.

Answer 1

A competitive inhibitor competes with the substrate for the active site of an enzyme, but a reaction usually does not occur once the inhibitor (I) is bound. While the inhibitor occupies the active site it prevents binding by the substrate. Competitive inhibitors are often compounds that resemble the substrate and combine with the enzyme to form an EI complex. This type of inhibition can be analyzed quantitatively by steady-state kinetics . Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the substrate simply by adding more substrate. Competitive inhibition is used therapeutically to treat patients who have ingested methanol, a solvent found in gas-line antifreeze. Methanol is converted to formaldehyde by the action of the enzyme alcohol dehydrogenase. Formaldehyde damages many tissues, and blindness is a common result because the eyes are particularly sensitive. Ethanol competes effectively with methanol as a substrate for alcohol dehydrogenase. The therapy for methanol poisoning is intravenous infusion of ethanol, which slows the formation of formaldehyde suff'iciently so that most of the methanol can be excreted harmlessly in the urine.

A noncompetitive inhibitor is one that binds to a site distinct from that which binds the substrate (Fig. 8-15); inhibitor binding does not block substrate binding (or vice versa). The enzyme is inactivated when inhibitor is bound, whether or not substrate is also present. The inhibitor effectively lowers the concentration of active enzyme and hence lowers the apparent V_max (V_max= K_cat[E_t]). There is often little or no effect on K_m. These characteristic effects of a noncompetitive inhibitor are further analyzed in Box 8-2. An uncompetitive inhibitor also binds at a site distinct from the substrate. However, an uncompetitive inhibitor will bind only to the ES complex. (The noncompetitive inhibitor binds to either free enzyme or the ES complex.

Irreversible inhibitors are those that combine with or destroy a functional group on the enzyme that is essential for its activity. Formation of a covalent link between an irreversible inhibitor and an enzyme is common. Irreversible inhibitors are very useful in studying reaction mechanisms. Amino acids with key catalytic functions in the active site can sometimes be identified by determining which amino acid is covalently linked to an inhibitor after the enzyme is inactivated.