# A New View of PAH Allostery - Correlation with Disease-Associated Alleles

> **NIH NIH R01** · RESEARCH INST OF FOX CHASE CAN CTR · 2020 · $397,851

## Abstract

PROJECT SUMMARY
Dysfunction of phenylalanine hydroxylase (PAH) is the most common inborn error of amino acid metabolism
and the underlying cause of phenylketonuria (PKU). By converting phenylalanine (Phe) to tyrosine, PAH
maintains blood Phe at levels sufficient for protein biosynthesis, but below neurotoxic levels. Regulation is
accomplished by allosteric activation by Phe. Based on extensive studies of individuals living with PKU, the
current medical consensus is to control blood Phe levels throughout life to achieve and maintain normal
neurological function; this argues for a better understanding of PAH structure/function relationships to support
both the understanding of existing pharmacological chaperones for PAH and the future development of novel
non-dietary therapeutics. In 2013 we introduced an innovative conformational selection model of PAH allostery
that includes a resting-state tetramer, an architecturally distinct activated tetramer, and smaller assemblies;
only activated PAH contains the allosteric Phe binding site. This site is at a multimer-specific subunit-subunit
interface, the details of which remain unknown. Our model includes a previously unforeseen domain rotation,
which is now strongly supported by recently published biophysical studies. 2016 marks our publication of the
first crystal structure for full length resting-state mammalian PAH; this is a long-awaited contribution to the field.
Small angle X-ray scattering (SAXS) supports both resting state PAH and Phe-stabilized activated PAH
tetramer structures, and confirms a major conformational difference between the two, which is consistent with
our allosteric model. The current application builds on these achievements. In AIM 1 we address the
relevance of our allosteric model to disease. We test whether specific common disease-associated PAH
variants are defective in the transition between resting-state and activated PAH and thus insensitive to
allosteric activation by Phe. This hypothesis is a major departure from the conventional view of PKU as a
protein folding/stability disorder. In AIM 2 we determine the structure of activated PAH using X-ray
crystallography and SAXS, and we extend our work with rat PAH to human PAH using a designed variant. In
AIM 3 we identify substances that can modulate PAH function (negatively or positively) by stabilizing either
resting-state or activated PAH. Using in vitro methods, we will screen approved drugs and environmental
contaminants, exposure to which can confound PKU phenotype. We use in silico screening of libraries of
drug-like molecules to provide leads for future development of new PKU therapies. All AIMS employ
established biochemical and biophysical methods to assess wild-type, disease-associated, and designed PAH
variants for the transition from resting to activated states. Key methods include intrinsic protein fluorescence,
SAXS, analytical ultracentrifugation, crystallography, native PAGE, enzyme kinetics, and the innovative...

## Key facts

- **NIH application ID:** 9981023
- **Project number:** 5R01NS100081-05
- **Recipient organization:** RESEARCH INST OF FOX CHASE CAN CTR
- **Principal Investigator:** EILEEN K JAFFE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $397,851
- **Award type:** 5
- **Project period:** 2016-09-15 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9981023

## Citation

> US National Institutes of Health, RePORTER application 9981023, A New View of PAH Allostery - Correlation with Disease-Associated Alleles (5R01NS100081-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9981023. Licensed CC0.

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