The activity of wild-type and mutant phenylalanine hydroxylase and its regulation by phenylalanine and tetrahydrobiopterin at physiological and pathological concentrations: An isothermal titration calorimetry study

Abstract

The activity of phenylalanine hydroxylase (PAH) is regulated by the levels of both the substrate ( l-Phe) and the natural cofactor (6 R)-tetrahydrobiopterin (BH 4). It has recently been observed that many PAH mutants associated with BH 4-responsive phenylketonuria display abnormal kinetic and regulatory properties as shown by standard kinetic analyses. In this work, we have developed a high-sensitive and high-throughput activity assay based on isothermal titration calorimetry (ITC) in order to study the kinetic properties of wild-type PAH (wt-PAH) and the BH 4-responsive c.204A > T (p.R68S) mutant at physiological and superphysiological concentrations of l-Phe and BH 4. Compared to wt-PAH, the p.R68S mutant showed reduced apparent and equilibrium binding affinity for the natural cofactor and increased affinity and non-cooperative response for l-Phe, together with a strong substrate inhibition that is alleviated at high BH 4 concentrations. For both wt-PAH and mutant, the apparent affinity for BH 4 decreases at increasing l-Phe concentrations, and the affinity for the substrate also depends on the cofactor concentration. Our results indicate that the activity landscape for wt and mutant enzymes is more complex than expected from standard kinetic analyses and highlight the applicability of this ITC-based assay to characterize the activity and regulation of PAH at a wide range of substrate and cofactor concentrations. Moreover, the results aid to understand the activity dynamics of wild-type and mutant PAH under physiological and pathological conditions, as well as BH 4-responsiveness in certain PKU mutations.