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.

FIGURE 1.

A novel continuous assay for the measurement of PAH activity. A, fluorescence intensity (I₃₀₄) of l-Tyr concentrations (0–150 μm) with increasing l-Phe concentrations (0, 243, 490, and 958 μm) is shown. l-Tyr fluorescence intensity was not influenced by increasing l-Phe concentrations, confirming spectral separation of the two substances in one mixed solution. Values are given as the mean ± S.E. of three independent measurements. B, quenching of l-Tyr fluorescence intensity by BH₄ is shown. Measurement of l-Tyr (0–150 μm) subsequent to the addition of increasing BH₄ concentrations (0, 25, 75, and 125 μm), revealed an inner filter effect of BH₄ on l-Tyr excitation and emission (left panel). For each BH₄ concentration used in the assay, a correction factor was calculated according to the factorial decrease in signal intensity to account for the inner filter effect (right panel). Values are given as the mean ± S.E. of three independent measurements. C, continuous measurement of time-dependent wild-type PAH kinetics with and without preincubation of the enzyme with 1 mm l-Phe are shown. D, l-Phe preincubation (activation) led to burst-phase kinetics within the first 30 s of the reaction (top) followed by a linear phase of l-Tyr production. Without l-Phe preincubation, an initial lag-phase before steady state enzyme kinetics was found (bottom, a red line was used to guide the eye). E, the time frame chosen for the measurement of steady state enzyme kinetics between 30 and 120 s showed a linear rate of reaction (top, with l-Phe preincubation; bottom, without l-Phe preincubation). F, a 96-well plate for sequential measurement of PAH enzyme kinetics is shown. Direct in-well measurements of enzyme kinetics of up to four different PAH enzymes (numbers 1–4) were performed by the sequential analysis of 2 rows, consisting of 24 wells (red box). Each row contained the PAH enzyme varying substrate concentrations (0–1 mm) and one cofactor concentration (75 μm) or varying cofactor concentrations (0–125 μm) and one substrate concentration (1 mm). Repeated cycles allowed kinetic measurements of 24 wells over a time period of 60 s.

FIGURE 2.

Measurements of wild-type PAH kinetics. A, reaction rates at variable l-Phe concentrations (0–1 mm) and one BH₄ concentration (75 μm) are shown. Before initiation of the reaction by BH₄, the enzyme was preincubated for 5 min at 25 °C with l-Phe to activate the enzyme. Nonlinear regression analysis was performed using the Hill equation. B and C, reaction rates at variable BH₄ concentrations (0–125 μm) and one l-Phe concentration (1 mm) are shown. B, data obtained for the l-Phe preincubated (activated) enzyme were evaluated using the Michaelis-Menten equation (dashed line) and the Hill equation (solid line). C, a comparison of enzyme kinetics measured using the non-activated (●) and the activated (○) wild-type PAH is shown. The non-activated enzyme showed non-cooperative binding of BH₄. The activated enzyme indicated positive cooperativity for the binding of BH₄. D, enzyme kinetics of non-activated and activated PAH at variable BH₄ concentrations (0–50 μm) and one l-Phe concentration (1 mm) are shown. Data obtained for the non-activated enzyme were fit to the Michaelis-Menten equation (left panel). Data of the activated enzyme followed Hill kinetics (right panel). For all enzyme activity measurements, fluorescence intensity was recorded and after subtraction of the blank reaction converted to enzyme activity units (nmol l-Tyr/min × mg protein) using the standard curve obtained by l-Tyr concentration measurements. Values are given as the mean ± S.E. of three independent experiments.

FIGURE 3.

Determining the activated structural and functional conformation. A, intrinsic tryptophan fluorescence emission spectra of the Factor Xa cleaved wild-type PAH, variant PAH R68S, and 103–427 are shown. Fluorescence emission spectra were acquired in the absence (dashed line) or presence (solid line) of 1 mm l-Phe. The excitation wavelength for Trp fluorescence measurements was 295 nm, with an excitation and emission slit of 2.5 and 5 nm, respectively. a.u., arbitrary units. B, differential scanning fluorimetry of the wild-type PAH and variant PAH R68S, V106A, and 103–427 fusion protein are shown. Denaturation of PAH was monitored by scanning a temperature range of 25 to 70 °C at a rate of 1.2 °C/min. Changes in 8-anilino-1-naphtalenesulfonic acid fluorescence emission were monitored at 500 nm (excitation 395 nm, slit widths 5.0/10.0 nm). The fraction unfolded of three to four independent experiments for wild-type, R68S, and 104–427 and seven to eight independent experiments for V106A without l-Phe (solid line) and with 1 mm l-Phe (dashed line) were determined (top panel; wild-type (black), R68S (blue), V106A (green), and 103–427 (red)), and the respective transition midpoints were calculated using the Boltzmann sigmoidal equation. For comparison of the transition midpoints, a paired t test, two-tailed, was used. Transition midpoints for wild-type and variant PAH, with (○) and without (●) l-Phe preincubation were plotted and compared (bottom panel) (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001). C, enzyme activity measured for the wild-type PAH and the preactivated variants R68S, V106A, and 103–427 without preincubation (●) and with preincubation of the enzyme (○) with 1 mm l-Phe before initiation of the reaction by the addition of BH₄. Data obtained for the non-preincubated and preincubated enzymes followed the Hill kinetic model as shown for the activated wild-type PAH. For all enzyme activity measurements, fluorescence intensity was recorded and after subtraction of the blank reaction converted to enzyme activity units (nmol of l-Tyr/min × mg protein) using the standard curve obtained by l-Tyr concentration measurements. Values are given as the mean ± S.E. of three independent experiments.