Chapter 1. Mirror Experiment Activity for Fig. 6.16: Do pancreatic enzymes function by binding to protein substrates?

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 shown in Fig. 6.16, 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 Fig. 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 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.