Activation of Phenylalanine Hydroxylase Induces Positive Cooperativity toward the Natural Cofactor

Abstract

Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ). Sapropterin dihydrochloride, the synthetic form of BH 4 , was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH 4 , a previously unknown finding. Analyses of structurally preactivated variants substantiated BH 4 -dependent cooperativity of the activated enzyme that does not rely on the presence of L-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.

A new model for allosteric regulation of phenylalanine hydroxylase: Implications for disease and therapeutics

“…PAH activity determination used a continuous fluorescence assay monitoring the production of tyrosine [57] adapted to single microcuvette format for use with a Photon Technologies International fluorescence spectrophotometer equipped with a xenon flash lamp. Fluorescence was monitored at λ Ex = 275 nm and λ Em = 305 nm, with excitation and emission slit widths set to 2 nm.…”

New protein structures provide an updated understanding of phenylketonuria

“…Here we abandon the concept of “misfolding with loss-of-function” and put a structural framework to prior proposals that disease-associated PAH variants can be defective in the allosteric response to elevated Phe in a manner that does not impair protein stability and/or promote aggregation (e.g. (21, 24)). On the basis of newly available structural information that defines the allosteric Phe binding site, we propose that there is a class of common disease-associated variants that are not defective in the ability to fold, but are defective in the ability to stabilize A-PAH.…”

Queuosine Deficiency in Eukaryotes Compromises Tyrosine Production through Increased Tetrahydrobiopterin Oxidation

“…A variety of other mechanisms are known to regulate PAH activity in vivo, including its activation by phenylalanine and phosphorylation, in addition to its allosteric inhibition by BH4 cofactor (31,32). Previous reports suggest that queuine can enhance the phosphorylation of unspecified cytosolic proteins (22), whereas in other cases a decrease in phosphorylation was observed (33)(34)(35).…”