Unmixing Enzyme Allostery

Abstract

Protein allostery is a key mechanism of metabolic control in all forms of life. Enzymes that change their activity in response to an effector are particularly important for regulating metabolite fluxes. I will discuss the application of small-angle X-ray scattering (SAXS) to study allosteric enzymes, which exist as complex mixtures in solution, using new strategies to “computationally purify” complex mixtures. In particular, I describe the powerful combination of chromatography-coupled SAXS and evolving factor analysis (EFA), an extension of singular value decomposition (SVD), which can separate scattering profiles of species that are not completely resolved by the column. We have used this technique to visualize the domain motions of phenylalanine hydroxylase (PheH), an enzyme that converts phenylalanine to tyrosine in the liver. The data support a model where tetrameric PheH is activated by dimerization of the regulatory domains, which bind phenylalanine in the active conformation. Surprisingly, the allosteric mechanism of this mammalian enzyme appears to be conserved from bacterial enzymes that synthesize aromatic amino acids.