Biomechanics of Phenylalanine Maintenance Illustrated in New Study

PHILADELPHIA (February 13, 2019) — Fox Chase Cancer Center researchers have published a groundbreaking study that describes for the first time the mechanisms of how the amount of amino acid phenylalanine (Phe) is controlled in blood. Accumulation of Phe can cause phenylketonuria (PKU), a rare genetic condition that can result in permanent neurological damage as well as behavioral abnormalities if not properly managed.

The paper, entitled, “Simulations of the regulatory ACT domain of human phenylalanine hydroxylase unveil its mechanism of phenylalanine binding,” appears in the December 2018 edition of The Journal of Biological Chemistry. It was highlighted as an Editor’s Pick for the issue, and was honored with an editorial piece that emphasized the work’s importance.

Phenylalanine hydroxylase (PAH) is an enzyme that regulates Phe levels to prevent neurotoxicity, which occurs in the most common genetic disorder of amino acid metabolism, PKU. Defects in the gene that encodes PAH is the cause of PKU. Doctors screen newborns for the disorder with a simple needle prick on the heel. People who have PKU must control the condition by following a strict diet that limits Phe, which is found in foods that contain protein or in Phe-containing compounds such as the artificial sweetener aspartame.

The researchers found out how PAH senses elevated Phe concentrations through transient allosteric Phe binding to a protein–protein interface between ACT domains of different subunits in a PAH tetramer.  This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. Allosteric Phe-binding was discovered to involve an unexpected gating motion that is required for binding and for release.  

“Our combined simulation and experimental study gives unprecedented insights into the conformational dynamics of the regulatory ACT domain of PAH and the molecular mechanism by which free Phe binds these domains,” wrote Eileen K. Jaffe, PhD, a professor at Fox Chase.

To investigate the sensing mechanism, Jaffe collaborated with Vincent Voelz, PhD, an associate professor of Chemistry at Temple University, and member of the Molecular Therapeutics program at Fox Chase. Voelz’s graduate student Yunhui Ge performed extensive molecular simulations on the Folding@home computing platform and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. The end result was a detailed mechanistic picture of how free Phe associates with the regulatory ACT domain dimer of PAH.

Structural intermediates identified from the simulations may be helpful in future studies of disease-associated mutations and provide new directions toward the development of PKU therapeutics.

Jaffe has pioneered the study of shape shifting proteins like PAH, whose normal physiological function requires major changes in protein assembly architecture, and for whom defects in these required shape changes contribute to disease. Although once thought to be anomalous, the basic science driving Jaffe’s work is now realized to be pertinent to proteins whose dysfunction causes cancer, and to provide a “shape-locking” approach to new therapeutics. 

This work was supported by National Institutes of Health Grants 1R01GM123296-01, 1S10OD020095-01, 1R01NS100081, and P30CA006927. It also received partial support from the National Science Foundation through Major Research Instrumentation Grant CNS-09-58854. The in vitro studies were also supported by the National PKU Alliance and BioMarin Pharmaceuticals.