Direct and continuous assay for prolyl 4-hydroxylase

Abstract

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in protocollagen strands. The resulting (2 S,4 R)-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 cosubstrates, 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 (2 S,4 S)-4-fluoroproline (flp), a proline analogue that is transformed into (2 S)-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 HPLC-based 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.