Conformational preferences of substrates for human prolyl 4-hydroxylase

Abstract

Prolyl 4-hydroxylase (P4H) catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in procollagen strands. The resulting (2 S,4 R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. Even though its product (Hyp) differs from its substrate (Pro) by only a single oxygen atom, no product inhibition has been observed for P4H. Here, we examine the basis for the binding and turnover of substrates by human P4H. Synthetic peptides containing (2 S,4 R)-4-fluoroproline (Flp), (2 S,4 S)-4-fluoroproline (flp), (2 S)-4-ketoproline (Kep), (2 S)-4-thiaproline (Thp), and 3,5-methanoproline (Mtp) were evaluated as substrates for P4H. Peptides containing Pro, flp, and Thp were found to be excellent substrates for P4H, forming Hyp, Kep, and (2 S,4 R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analogue that does bind to P4H is also a substrate, indicating that discrimination occurs at the level of binding rather than turnover. As the iron(IV)-oxo species that forms in the active site of P4H is highly reactive, P4H has an imperative for forming a snug complex with its substrate and appears to do so. Most notably, those proline analogues with a greater preference for a C (gamma)- endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C (gamma)- exo pucker and trans peptide bond, P4H appears to discriminate against the conformation of proline residues in a manner that diminishes product inhibition during collagen biosynthesis.

Figure 1

Reaction catalyzed by prolyl 4-hydroxylase.

Figure 2

Structures of prolines and its analogs studied herein, along with their predominant ring pucker. Each amino acid was incorporated into a tetrapeptide and examined as a substrate or inhibitor of P4H.

Figure 3

Comparison of PEG-Gly–Tyr–Yaa–GlyOEt peptides, where Yaa = Pro (●), flp (○), or Thp (▼;), as substrates of P4H. Plot shows the rate of product formation at varying substrate concentrations. Reactions were performed as described in the Experimental Methods section. Reactions were initiated by the addition of tetrapeptide substrate (0.05–3 mM), and run at 30 °C for 5 min. Product formation was analyzed by HPLC. Individual points are the average (± SE) of three independent reactions. Data were fitted to the Michaelis–Menten equation to obtain kinetic parameters.

Figure 4

Peptides containing Flp, Kep, or Mtp do not inhibit Hyp formation from Pro by P4H. Graph shows the hydroxyproline formed from proline in the presence of no proline analog, Kep, Flp, or Mtp. The reaction with no proline analog was designated as 100%. Reactions contained 0.6 mM PEG-Gly–Tyr–Yaa–GlyOEt, where Yaa = Kep, Flp or Mtp, and 0.06 mM PEG-Gly–Tyr–Pro–GlyOEt. Individual points are the average (± SE) of three independent reactions.

Figure 5

Characterization of the products for the turnover of PEG-Gly–Tyr–flp–GlyOEt by P4H. (A) Kep (2) is formed from flp (1) by P4H. Kep is reduced with sodium borohydride (10 equiv) for 30 min to form Hyp and hyp (3, 4), or Kep was converted to an oxime (5) by reaction with hydroxylamine (10 equiv) in 250 mM sodium phosphate buffer, pH 5.0, for 1 h at 100 °C. (B) HPLC analysis of PEG-Gly–Tyr–flp–GlyOEt reactions. (i) Turnover by P4H; (ii) turnover by P4H and treatment with sodium borohydride; (iii) turnover by P4H and treatment with hydroxylamine. Peak 1,2 is from the coeluting peptides containing flp or Kep; peaks 3 and 4 are from the peptides containing Hyp or hyp; peak 5 is from the peptide of containing the oxime of Kep. HPLC conditions are described in the Experimental Procedures section.

Figure 6

Characterization of the products for the turnover of PEG-Gly–Tyr–Thp–GlyOEt by P4H. (A) Thp(O) (2) is formed from Thp (1) upon catalysis by P4H. Thp(O) and thp(O) (2,3) are formed from Thp upon reaction with MCPBA (1 equiv) in chloroform for 2.5 h. Thp(O,O) (4) is formed from Thp upon reaction with MCPBA (10 equiv) in chloroform for 5 h. (B) HPLC analysis of reactions of PEG-Gly–Tyr–Thp–GlyOEt. (i) Turnover by P4H; (ii) treatment with MCPBA (1 equiv); (iii) treatment with MCPBA (10 equiv). Peak 1 is from the peptide containing Thp peptide; peaks 2 and 3 are for the peptides containing Thp(O) or thp(O); peak 4 is for the peptide containing Thp(O,O). HPLC conditions are described in the Experimental Procedures section.

Figure 7

Putative model for substrate recognition by P4H. The Pro residue of the substrate has a cis peptide bond and C^γ-endo ring pucker; the iron(IV)-oxo speices is in a position to remove the proR hydrogen on C-4 (31). Hydroxylation changes the preferred conformation, and the Hyp residue of the product has a trans peptide bond and C^γ-exo ring pucker. The structures of Pro and Hyp are actual fragments of the crystal structures of AcProOMe and AcHypOMe (76), and are aligned such that the nitrogen and its three pendant carbons are in spatial alignment.

Scheme 1