Direct and continuous assay for prolyl 4-hydroxylase

Abstract

Prolyl 4-hydroxylase (P4H) is a non-heme iron dioxygenase that catalyzes the post-translational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O 2 as co-substrates, and forms succinate and CO 2 as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLCbased assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.Collagens are the major structural proteins of the extracellular matrix. Collagens undergo a number of post-translational modifications during biosynthesis. One major modification is the hydroxylation of certain prolyl residues. The repeating amino-acid sequence of collagen, wherein every third residue is a glycine (Gly): Xaa-Yaa-Gly, is rich in (2S)-proline (Pro). Indeed, Pro is the most common amino acid found in the Xaa position [1]. In the Yaa position, the most common amino is (2S, 4R)-4-hydroxyproline (Hyp). Hyp is formed by a posttranslational modification of Pro catalyzed by prolyl 4-hydroxylase (P4H; EC 1.14.11.2).Hyp is necessary for the stable formation of the triple-helical structure of collagen. Collagen with decreased levels of Hyp is defective in folding and secretion under physiological conditions [2][3][4]. P4H activity is required for the viability of the nematode Caenorhabditis elegans [5,6] and the mouse Mus musculus [7]. In vitro, P4H has been studied by availing enzyme via heterologous expression in insect cells, yeast, and (only recently [8,9]) bacteria. *Corresponding author. Tel: 608-262-8588. Fax: 608-890-2583. E-mail address: rtraines@wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the conten...

4-Fluoroprolines: Conformational Analysis and Effects on the Stability and Folding of Peptides and Proteins

“…Enzymatic discrimination during substrate binding and not during turnover was evident from the hydroxylation of peptides bearing flp and the absence of binding to peptide counterparts containing Flp residues. Oxidation of flp to 4-ketoproline (Kep) by P4H was used to develop a probe of enzymatic activity by measuring fluoride-ion release in a continuous assay [78]. Shedding light on the mechanism of collagen hydroxylation, these studies have provided information for designing inhibitors of P4H to treat fibrotic diseases associated with an overabundance of collagen.…”

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

“…Higher salt concentrations (500 mM) decreased P4H activity by ~80%. The D414A variant of P4H, which has an active site analogous to that of SyrB2, was produced, and, as expected [16], lost its hydroxylase activity. Still, no halogenated product from reaction mixtures containing D414A P4H was detected by high-performance liquid chromatography (HPLC) or mass spectrometry, even in the presence of 100 mM sodium fluoride, chloride, or bromide (Table 1).…”

“…The ability of P4H to catalyze the hydroxylation or halogenation of a proline residue was monitored with an HPLC-based assay described previously [16], [26], [27]. Assays were conducted for 20 min at 30°C in 100 µL of 50 mM TrisHCl buffer, pH 7.8, containing dithiothreitol (100 µM), catalase (100 µg/mL), ascorbate (2 mM), FeSO4 (50-300 µM), α-ketoglutarate (0.5-25 mM), bovine serum albumin (1.0 mg/mL), and P4H (0.09-1.5 µM).…”

Prolyl 4-Hydroxylase: Substrate Isosteres in Which an (E)- or (Z)-Alkene Replaces the Prolyl Peptide Bond

“…(B) Putative enzymic ligands for CP4H. 15–17 (C) Proline analogs that are substrates for CP4H, 30–33 despite substitutions at C β , C γ ( i.e. , C4), or C δ .…”