The influence of allosteric activation on the active site properties of phenylalanine hydroxylase

• Main Author: Nolan, Douglas
• Other Authors: Caradonna, John P.
• Language: en_US
• Published: 2021
• Subjects: Biochemistry
• Online Access: https://hdl.handle.net/2144/42023

Phenylalanine Hydroxylase (PheH, EC: 1.14.16.1) is a non-heme iron monooxygenase that converts L-Phe to L-Tyr using a tetrahydrobiopterin coenzyme and dioxygen. PheH is allosterically regulated by its substrate L-Phe. Binding of L-Phe to the allosteric site causes PheH to convert from a low-activity T-state to a high-activity R-state. Upon the T to R conversion, PheH experiences an increase in hydrophobicity, a shift in fluorescence emission peak maximum, increased hydrodynamic radius, and elongation of its quaternary structure. While PheH has been well characterized in terms of these global changes that occur upon allosteric activation, less is known about how the active site properties change upon this process. Herein, we report the use of cw-EPR and pulsed-EPR techniques (electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) methods) to probe the properties of the active site of PheH with respect to the primary coordination sphere of the non-heme iron center and the orientation of pterin coenzyme and substrate L-Phe. A suit of cw-EPR experiments using 17O enriched water and 1H-HYSCORE approaches were used to probe the water coordination of the iron center across mechanistic states of PheH and select mutants. The ESEEM technique was combined with 2H labelled L-Phe and H4pterin coenzyme to examine how the binding orientation of both L-Phe and H4pterin change upon allosteric activation. These results demonstrate that L-Phe allosteric activation causes the pterin coenzyme to move ~1.8 Å closer to the iron center. Furthermore, 2H-ESEEM samples prepared at non-activating concentrations of L-Phe suggest that its binding in the active site alone is not sufficient to induce the movement of the pterin coenzyme. 2H-ESEEM studies using site specifically deuterated L-Phe demonstrate that allosteric activation and binding of pterin cause the reorientation of L-Phe such that both the meta and ortho positions are similarly close to the iron center. Lastly, steady state kinetic measurements of pterin oxidation within the active site of apo-wtPheH and apo-PheH118-452 were measured to understand how the active site influences the reactivity of pterin with O2. These results will be explained in the context of the regulatory properties of PheH as well as the catalytic mechanism.