Chapter 1. Mirror Experiment Activity 6.17

Experiment

Background

You may know the pancreas as the site of insulin secretion, but this organ also produces enzymes that play a role in digestion. As you have learned in Figures 6.17 and 6.18, spectral analyses and radiolabeling experiments have demonstrated that enzymes can bind to substrates; however, this work yielded few clues to the physical shape of the enzyme-substrate complexes that were studied. Do pancreatic enzymes also function by binding to protein substrates? If so, can researchers determine the shape of pancreatic enzyme-substrate complexes?

Hypothesis

Edgar Meyer and colleagues hypothesized that pancreatic enzymes are able to form complexes with protein substrates. They also sought to determine the three-dimensional conformation of the enzyme-substrate complexes formed by these enzymes.

Experiment

Researchers often use one of two methods to determine the shape of a protein: X-ray crystallography or nuclear magnetic resonance (NMR). As discussed in Figure 4.5, scientists using X-ray crystallography first produce a crystal of their target protein, then expose this crystal to X-rays. The crystalized protein generates a specific pattern of reflected X-rays, which can be visualized on film (in much the same way as your bones can be visualized in an X-ray). NMR is carried out in a similar manner although the protein does not have to be in crystalline form; however, the protein is exposed to magnetic fields instead of X-rays. In both cases, data collected from X-ray crystallography and NMR experiments help researchers predict the three-dimensional shape of a protein.

Edgar Meyer and colleagues chose to study pig elastase, an enzyme produced by the pancreas; they evaluated the ability of this enzyme to bind to a hexapeptide substrate, a short molecule composed of six amino acids. In their first experiment, an elastase crystal was generated and submerged in a hexapeptide solution; X-ray crystallography was performed on the crystal. In their second experiment, solutions of uncrystalized hexapeptide and uncrystalized elastase were mixed together and NMR was carried out (Figure 1).

Results

Using both X-ray crystallography and NMR techniques, Meyer and colleagues determined that elastase is able to bind a hexapeptide substrate. Furthermore, researchers were able to visualize the three-dimensional structure of this elastase-hexapeptide complex.

Source

Meyer, E. F., Jr., et al., 1988. Analysis of an enzyme-substrate complex by X-ray crystallography and transferred nuclear Overhauser enhancement measurements: porcine pancreatic elastase and a hexapeptide. Biochemistry. 27, 725-30.