SEC, SAXS and informatics uncover secrets of liver enzyme

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  • Published: May 31, 2016
  • Author: Jon Evans
  • Source: Princeton University
  • Channels: HPLC / Laboratory Informatics / X-ray Spectrometry / Chemometrics & Informatics

Image showing SEC-SAXS data (with cyan cross-section showing the elution profile and magenta cross-section showing scattering profile) and the structure of the activated phenylalanine hydroxylase.

Image: Ando lab.

Using a powerful combination of analytical techniques, including size exclusion chromatography and x-ray scattering, researchers have revealed new insights into the mechanism of a liver enzyme that is critical for human health. The enzyme, phenylalanine hydroxylase, turns the essential amino acid phenylalanine, which is found in eggs, beef and many other foods and as an additive in diet soda, into tyrosine, a precursor for many important neurotransmitters.

"We need phenylalanine hydroxylase to control levels of phenylalanine in the blood because too much is toxic to the brain," explained Steve Meisburger, a post-doctoral researcher at Princeton University in New Jersey, US, and lead author of a paper reporting this research in the Journal of the American Chemical Society. Genetic mutations in phenylalanine hydroxylase can lead to disorders such as phenylketonuria, an inherited condition that can cause intellectual and behavioral disabilities unless detected at birth and managed through a strict diet.

The researchers have now been able to uncover structural data on phenylalanine hydroxylase's active state that has eluded scientists for years. "It's a floppy enzyme which means it's dynamic," said Nozomi Ando, an assistant professor of chemistry at Princeton and corresponding author of the paper. "That also means it doesn't like to crystallize," she added. This obviously creates problems for studying the enzyme with x-ray crystallography, which requires solid crystal samples. Although efforts to crystallize phenylalanine hydroxylase have recently met with success, they have still only captured the enzyme in its inactive state.

Meisbruger, Ando and their colleagues were able to bypass the tricky challenge of growing crystals of the active enzyme by using their expertise in small angle x-ray scattering (SAXS), which can study enzymes in a solution. And because the enzyme is susceptible to aggregation or clumping up in solution, the researchers coupled SAXS with size exclusion chromatography (SEC).

"Pairing SEC with SAXS is an emergent technique. Our contribution is that we saw a clever way to use it," Ando explained. To conduct SAXS on the separated enzyme, the researchers took advantage of the powerful x-rays produced by the Cornell High Energy Synchrotron Source in Ithaca, New York. "Any time on the machine that is available, we use it. Not a single photon gets wasted," Ando said. The resulting dataset is quite complex as the sample also contains phenylalanine, so that the researchers can catch the enzyme in action.

"Current approaches for analyzing this type of dataset are very crude," Meisburger said, because they assume that each peak represents a single species, whereas in this case each peak is actually a mixture of species. So the team used an advanced linear algebra method known as evolving factor analysis that allowed them to separate the scattering components. "We can use these linear algebra methods to 'un-mix' species that are overlapping," Meisburger said, "That's the piece that I think is really exciting."

By applying their unique approach, the researchers were able to provide experimental support for a model of the active structure of phenylalanine hydroxylase that builds upon recent work by their collaborators at the University of Texas Health Science Center at San Antonio. In this model, two phenylalanine molecules dock to a pair of sites on the enzyme, bringing a pair of arms together and freeing up the active sites when more phenylalanine molecules come along.

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