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.
Highlights
(BayGene). □S The on-line version of this article contains supplemental Materials and Methods, Tables 1 and 2, and Figs. 1 and 2. 1 These authors contributed to this work. 2 Part of an M.D. thesis to be submitted at Ludwig-Maximilians-University, Munich, Germany. 3 To whom correspondence should be addressed: Lindwurmstrasse 4, 80337
Analyses of the effects of missense mutations in the phenylalanine hydroxylase (PAH) gene on PAH enzyme kinetic properties have shown that residual enzyme activity generally is high, yet allostery is often disturbed [17,18,19], with reduced cooperativity for substrate binding, decreased substrate activation, or altered affinity to the substrate and the cofactor
Evaluation of the Inner Filter Effect (IFE) of BH4 and calculation of the correction factor for each BH4 concentration added to the reaction mixture was performed before each enzyme kinetic measurement (Fig. 1B)
Summary
(BayGene). □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Materials and Methods, Tables 1 and 2, and Figs. 1 and 2. 1 These authors contributed to this work. 2 Part of an M.D. thesis to be submitted at Ludwig-Maximilians-University, Munich, Germany. 3 To whom correspondence should be addressed: Lindwurmstrasse 4, 80337. Binding of the substrate induces a catalytically competent (activated) enzyme, whereas binding of BH4 leads to formation of an inactive dead-end PAH-BH4 complex (9 –12) These regulatory mechanisms require reversible conformational changes that are transmitted throughout the enzyme upon binding of BH4 and L-Phe [13]. The evaluation of kinetics and allostery can help to assess to which extent local single amino acid replacements lead to global conformational alterations compromising enzyme function. In this context, comprehensive steady state kinetic analyses beyond single determination of residual enzyme activity are needed to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Cofactor-dependent kinetic studies were routinely performed using the non-activated PAH enzyme [19, 26], whereas an L-Phe preincubated (activated) enzyme was applied in our experiments
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