Investigating PAH Equilibrium Shift in Conformational Flexibility

Saving Patient Mutations Causing PKU

Project by Alexander Kang
Gupta-Van Duyne Lab, University of Pennsylvania Perelman School of Medicine

- Phenylketonuria (PKU) is an autosomal recessive metabolic disorder affecting

~1:10,000 live births
- Seizures, hyperactivity, tremor, stunted growth, albinism
- Main treatment is diet

Dopamine, Thyroxine,
Norepinephrine, epinephrine,
melanin

Serotonin, Melatonin, tryptamine,

vitamins and cofactors (niacin, NAD,
Vrana, K. How the regulatory and catalytic domains get together. NADP)
Nat Struct Mol Biol 6, 401–402 (1999). https://doi.org/10.1038/8192
What We Know:
- Phe80 in rat PAH anchors the
autoinhibited conformation
- Tested F80D, F80L, F80A, and
F80R with activity assays, IEC,
and limited proteolysis

Activated State (A-PAH)

Resting State (RS-PAH)
• High Activity
• Low Activity (50-120 µM PHE)
• ACT Domain Dimer via dramatic
• Autoinhibited
conformational change
• ACT Domains occlude active site
• Active sites accessible
• No allosteric PHE binding site
• PHE binding site in ACT dimer
• Atomic Structures (MX, EM)
• No Atomic Model
 Methods: Inverse PCR Mutagenesis

- Investigate whether
F80R can save patient
mutations by
continuing to shift
the equilibrium to
the activated state
- Patient Mutations
Tested: R68S (RD),
R158Q (CD), R261Q
(CD), R408W (OD)
Data & Future Endeavors:

R68S Mutations
PCR Reactions on
0.75% Agarose Gel

5den Mammalian PAH Tetramer

12.5% SDS PAGE Gel for PAH

Project (first four are pre-induction,
rest are post-induction alternating PyMOL
WC and S for WT R68S, F80R
Molecular
R68S, and WT)
Visualization
Software