Observing Single Enzymes At Work | October 29, 2007 Issue - Vol. 85 Issue 44 | Chemical & Engineering News
Volume 85 Issue 44 | p. 8 | News of The Week
Issue Date: October 29, 2007

Observing Single Enzymes At Work

Method captures the interaction between a single enzyme molecule and its inhibitor
Department: Education
News Channels: Analytical SCENE

THE ABILITY TO OBSERVE the binding and release of an inhibitor to a single enzyme molecule opens many doors. It can uncover mechanistic information that is masked by analysis of bulk solutions. It may also help identify inhibitors as potential drugs. Researchers at Tufts University report having made such observations for the first time (Proc. Natl. Acad. Sci. USA, DOI: 1073/pnas.0705411104).

To enable such an intimate look at enzymes, chemistry professor David R. Walt and postdocs Hans H. Gorris and David M. Rissin trap thousands of individual enzyme molecules in separate, tiny reaction chambers, or wells, each one etched onto an end of a glass optical fiber in a large bundle. Along with the single enzyme molecule in the well are lots of the enzyme's substrate and a small amount of an enzyme inhibitor. When the fiber bundle is mounted on a fluorescence microscope, fluorescent molecules released by the reaction can be detected.

Walt and coworkers applied the technique to a study of the time-dependent interactions between the enzyme β-galactosidase, a substrate analog called resorufin-β-galactopyranoside, and the inhibitor D-galactal. When hydrolyzed by the enzyme, the substrate releases the fluorescent compound resorufin.

β-Galactosidase has four identical subunits, and the kinetics and mechanism of inhibition depend on whether binding at one subunit affects binding at the others. If the four catalytic sites are independent, the enzyme's activity should change in steps as the number of inhibitor molecules bound changes from zero (enzyme completely on) to four (completely off). But if one site affects the others, a phenomenon called cooperative binding, only two states—on and off—would exist.

With D-galactal as the inhibitor, β-galactosidase appears to toggle between on and off. "It's pretty likely that this particular tetrameric enzyme works with this inhibitor via a cooperative mechanism of binding and release," Walt says.

Norman J. Dovichi, a chemistry professor at the University of Washington, Seattle, who also studies individual enzymes, calls the evidence for cooperative inhibitor binding a "quite impressive result."

The method can be used on a wide range of enzymes to identify inhibitors as potential drugs, Walt says. "We can measure kinetic constants and binding constants of inhibitor molecules with enzymes using incredibly small amounts of material."

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