The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP

Abstract

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity.

FIGURE 1.

Comparison of enzymatic characteristics of recombinant rat wt-GTPCH and Δ45-GTPCH. Activity of 0.1 μm wt-GTPCH or Δ45-GTPCH was assayed in 100 mm Tris-HCl (pH 7.8) at 37 °C with 5–5000 μm GTP, using the kinetic microplate assay as described under “Experimental Procedures.” Shown is a typical plot of reaction rate versus substrate concentration from a single experiment. Values are mean ± S.E. of triplicate measurements. Nonlinear regression analysis was used to fit a curve to the data points; summary kinetic parameters from this and other experiments are provided under supplemental Table S3.

FIGURE 2.

Comparison of the effects of His₆-GFRP on wt-GTPCH and Δ45-GTPCH activity. GTPCH assays were conducted in the presence or absence of His₆-GFRP, in 100 mm Tris-HCl (pH 7.8) at 37 °C, using the kinetic microplate assay described under “Experimental Procedures.” A, 0.1 μm wt-GTPCH or Δ45-GTPCH activity was assayed in the absence or presence of 1 μm His₆-GFRP in reactions containing 5–500 μm GTP. Shown is a typical plot of the reaction rate versus substrate concentration from a single experiment. Values are mean ± S.E. of triplicate measurements. Nonlinear regression analysis was used to fit a curve to the data points. B, the activity of 0.1 μm wt-GTPCH and Δ45-GTPCH were assayed in the absence or presence of either 1.0 or 0.1 μm His₆-GFRP, in reactions containing 1 mm GTP. Values are mean ± S.E. of quintuplicate measurements, and compared by analysis of variance. ***, p < 0.001; **, p < 0.01. ns, not significant.

FIGURE 3.

Native complex formation between His₆-GFRP and wt-GTPCH or Δ45-GTPCH. wt-GTPCH or Δ45-GTPCH (2.5 μm) were incubated in 100 mm Tris-HCl (pH 7.8) at 37 °C for 1 h. In some reactions, 1 mm GTP and/or 2.5 μm His₆-GFRP were also included. A, mixtures containing wt-GTPCH (67.5 ng) or Δ45-GTPCH (55.3 ng) and His₆-GFRP (29.5 ng) were resolved on a native PAGE BisTris 4–20% gel before transfer to PVDF membrane and Western blotting (IB) with affinity purified antiserum to GFRP. Black arrows highlight laddering complexes. B, the same PVDF membrane as in A, after stripping and reprobing with antiserum to GTPCH. Unlabeled arrows are as in A, to indicate the position of species identified with anti-GFRP antiserum.

FIGURE 4.

His₆-GFRP-mediated allosteric regulation of GTPCH by small molecules. A, the activity of 0.1 μm wt-GTPCH or Δ45-GTPCH was assayed in the presence or absence of 1 μm His₆-GFRP in 100 mm Tris-HCl and 3 mm Phe (pH 7.8) at 37 °C in reactions containing 5–500 μm GTP, using the kinetic microplate assay described under “Experimental Procedures.” Shown is a typical plot of reaction rate versus substrate concentration from one experiment. Values plotted are mean ± S.E. of triplicate measurements. B, the activity of 0.1 μm wt-GTPCH or Δ45-GTPCH was assayed in 1.5 mm DAHP or 1.5 mm DAHP and 3 mm Phe in 100 mm Tris-HCl (pH 7.8) at 37 °C, 1 mm GTP, in reactions containing 0.01 to 1 μm His₆-GFRP, using the kinetic microplate assay described under “Experimental Procedures.” Values are plotted as a percentage of GTPCH activity, measured in the absence of His₆-GFRP. Shown is a typical plot of reaction rate versus His₆-GFRP concentration from a single experiment. Values are mean ± S.E. of triplicate measurements. Nonlinear regression analysis was used to fit the solid curve to the data points, whereas a dotted line simply connects all points.

FIGURE 5.

Effect of a synthetic GTPCH-(2–45) N-terminal peptide (N45) on activity of GTPCH and regulation by His₆-GFRP. The activity of 0.1 μm Δ45-GTPCH was assayed in 100 mm Tris-HCl (pH 7.8) at 37 °C, 1 mm GTP, in reactions containing 0–30 μm N45 peptide and 1 μm His₆-GFRP. A, the N45 peptide corresponds to the N-terminal 44 amino acids of rat GTPCH (i.e. GTPCH-(1–45), minus the initial methionine residue). B, Δ45-GTPCH activity in the absence or presence of His₆-GFRP, plotted as a function of peptide concentration. Values are mean ± S.E. of quintuplicate measurements. C, percent stimulation of 0.1 μm wt-GTPCH or Δ45-GTPCH activity by 1 μm His₆-GFRP in the absence or presence of 30 μm N45 peptide. Values are mean ± S.E. of quintuplicate measurements. Analysis by one-way analysis of variance was used, **, p < 0.01. ns, not significant; p, > 0.05.

FIGURE 6.

Comparison of biopterin accumulation in HEK-293 cells after transfection with wt-GTPCH or Δ45-GTPCH, normalized for GTPCH expression levels. HEK293 cells were transiently transfected with pcDNA3-based plasmid vectors that express either wt-GTPCH or Δ45-GTPCH. Cells were harvested after 24 h of protein expression and levels of biopterin (BH2 + BH4) in cell lysates were quantified based on fluorescence, following HPLC separation and electrochemical oxidation (see “Experimental Procedures” for details). Enzymatic activity was normalized to the amount of wt-GTPCH or Δ45-GTPCH, as determined by quantitative densitometry after Western blotting. Values were compared by t test, *, p < 0.05.