Communications to the Editor Mechanism-Based Inactivation of the Human Prolyl-4-hydroxylase by 5-Oxaproline-Containing Peptides: Evidence for a Prolyl Radical Intermediate Min Wu, Hong-Sik Moon, and Tadhg P. Begley* Department of Chemistry and Chemical Biology Cornell UniVersity, Ithaca, New York 14853 Johanna Myllyharju and Kari I. Kivirikko Collagen Research Unit Biocenter and Department of Medical Biochemistry UniVersity of Oulu, Kajaanintie 52A FIN-90220, Oulu, Finland ReceiVed April 9, 1998 Prolyl-4-hydroxylase catalyzes the hydroxylation of proline residues at X-Pro-Gly sequences in procollagen (Scheme 1). This reaction is an essential step in the biosynthesis of collagen, the major protein component of connective tissue. Prolyl-4-hydroxylase (human) is an R 2  2 tetramer (R) 59 000 Da,  ) 55 000 Da) and requires Fe(II), R-ketoglutarate, oxygen, and ascorbate for activity. 1 The genes for the R and  subunits of the human enzyme have been cloned, sequenced, and over- expressed in a baculovirus expression system, and the enzyme can be readily purified in multi-milligram quantities. 2 5-Oxaproline-containing peptides have been previously identi- fied as mechanism-based inactivating agents for prolyl-4-hy- droxylase. 3 The mechanism of this inactivation has not been determined. As a first step toward elucidating this mechanism, we report here the synthesis of a highly fluorescent 5-oxaproline- containing peptide 5 and the identification of its prolyl-4- hydroxylase-catalyzed oxidation product. Two mechanisms for the enzymatic oxidation of 5 were considered (Scheme 2). In mechanism A, hydrogen-atom abstrac- tion from the oxaproline by the active site ferryl [Fe IV ) O] intermediate would give radical 7. Recombination followed by product dissociation would give the hemiacetal 9. In mechanism B, -scission of the weak NO bond of 7 would give 10. Addition of the iron(III)hydroxide to the aldehyde followed by intra- molecular hydrogen-atom transfer 4 and product dissociation would give 13 in which the oxaproline moiety has been oxidized to aspartic acid. Peptide 5 was synthesized as outlined in Scheme 3. 5,6 This was an efficient suicide substrate for prolyl-4-hydroxylase, covalently labeling the enzyme, and only trace quantities of a polar reaction product could be detected in the reaction mixture. 7,8 This product was purified, 9 and FAB-MS analysis demonstrated that the enzymatic oxidation resulted in the addition of one atom ofoxygen to 5. 10 This is consistent with the formation of either 9 or 13. Peptides 9 and 13 were synthesized to differentiate between mechanisms A and B. Chromatographic comparison of these peptides with the enzymatic product demonstrated that 9 was not formed and that the only enzymatic product was 13. This was confirmed by scale-up of the enzymatic reaction and the isolation of the reaction product in sufficient quantities for complete spectroscopic characterization ( 1 H NMR, COSY, HMQC, HMBC, HRFAB-MS, and MS-MS). The spectra of the enzymatic product and the spectra of peptide 13 were identical. While the R-ketoglutarate-dependent monooxygenases are not as well-studied as the heme-dependent monooxygenases, several (1) (a) Kivirikko, K. I.; Pihlajaniemi, T. AdV. Enzymol. Relat. Areas Mol. Biol. 1998, 72, 325. (b) Kivirikko, K. I.; Myllyla, R.; Pihlajaniemi, T. In Posttranslational Modification of Proteins; Harding, J., Crabbe, M. Eds.; CRC Press: Boca Raton, FL, 1992; Chapter 1. (2) Vuori, K.; Pihlajaniemi, T.; Marttila, M.; Kivirikko, K. I. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7467. (3) Gunzler, V.; Brocks, D.; Henke, S.; Myllyla, R.; Geiger, R.; Kivirikko, K. I. J. Biol. Chem. 1988, 263, 19498. (4) Protein-mediated hydrogen-atom transfer is also possible. (5) Vasella, A. HelV. Chim. Acta. 1977, 60, 426. (6) (a) Vasella, A.; Voeffrey, R. J. Chem. Soc., Chem. Commun. 1981, 97. (b) Vasella, A.; Voeffrey, R.; Pless, J.; Huguenin, R. HelV. Chim. Acta. 1983, 66, 1241. (7) The enzymatic reaction mixture consisted of peptide 5 (0.85 mM), FeSO4 (0.05 mM), ascorbic acid (2.0 mM), BSA (1 mg), catalase (0.05 mg), DTT (0.1 mM), R-ketoglutarate (0.5 mM) Tris-HCl (50 mM, pH ) 7.8) prolyl- 4-hydroxylase (68 µg) in 500 µL. Compound 13 was not formed in control reactions from which the enzyme or R-ketoglutarate were excluded. (8) The inactivation of prolyl-4-hydroxylase by 5 follows nonpseudo-first- order kinetics due to consumption of the inhibitor. We have estimated the rate constant for the inactivation at several inhibitor concentrations (0, 0.5, 1, 1.5, 2, and 5 µM) by determining the activity remaining after two minutes using the previously described assay procedure. 3 From these data, we determine that kinact ) 0.6 min -1 and KI ) 1.6 µM. The enzyme is protected from inactivation by the substrate (PPG)10. To demonstrate covalent labeling of the enzyme, we have synthesized an analogue of peptide 5 in which the ethoxy group of the ester has been replaced with biotin hydrazide. This also inactivates the enzyme, and we have demonstrated stable covalent attachment of the label to the enzyme (both subunits) by SDS PAGE followed by western blotting and visualization of biotin-labeled protein with horseradish peroxidase conjugated streptavidin and the Pierce SuperSignal substrate. Scheme 1 Scheme 2 587 J. Am. Chem. Soc. 1999, 121, 587-588 10.1021/ja981193h CCC: $18.00 © 1999 American Chemical Society Published on Web 01/06/1999