Conformational Preferences of Substrates for Human Prolyl 4-Hydroxylase

Abstract

Prolyl 4-hydroxylase (P4H) catalyzes the post-translational hydroxylation of (2S)-proline (Pro) residues in procollagen strands. The resulting (2S,4R)-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 (2S,4R)-4-fluoroproline (Flp), (2S,4S)-4-fluoroproline (flp), (2S)-4-ketoproline (Kep), (2S)-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 (2S,4R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analog 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 analogs with a greater preference for a C γ -endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C γ -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.Collagens are the major structural proteins in the extracellular matrix. All collagens are comprised of three polypeptide strands that coil together into a right-handed triple helix. Each strand contains a repeating three amino-acid sequence in which every third residue is a glycine (Gly 1 ): Xaa-Yaa-Gly. The Xaa position is often (2S)-proline (Pro), and the Yaa position is often (2S,4R)-4-hydroxyproline (Hyp) (1). Hyp is produced by the action of prolyl 4-hydroxylase (P4H; EC 1.14.11.2). This post-translational modification is the most prevalent † This work was supported by Grants AR044276 (NIH to R.T.R) and CHE 0515635 (NSF to G.R.K.). K.L.G. was supported by ChemistryBiology Interface Training Grant T32 BM008505 (NIH). MALDI-MS experiments were performed at the University of WisconsinMadison Biophysics Instrumentation Facility, which was established with Grants BIR-9512577 (NSF) and RR13790 (NIH).*To whom correspondence should be addressed: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544. Telephone: 608-262-8588. Fax: 608-262-3453. rtraines@wisc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 22. in the kingdom Animalia, where Hyp is more common than seven of the "common" aminoacid residues: Cys, Gln, His, Met, P...

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

“…Proline derivatives such as flp, which prefer relatively the endo pucker and the cis peptide bond conformation proved better P4H substrates [77]. 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.…”

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

“…These data are consistent with a previous correlation indicating that substrates for human CP4H1 tend to have greater preference for a cis prolyl peptide bond. 32 In contrast to isosteres 3–6 , however, none of those previous substrates were fixed as a cis (or trans ) isomer. Due to more reliable quantitation and sensitivity compared to the Cbz-peptides, we proceeded with the dansyl isosteres 3 and 5 in subsequent analyses.…”

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

“…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).…”