Mechanism-based inactivation of the human prolyl-4-hydroxylase by 5- oxaproline-containing peptides: Evidence for a prolyl radical intermediate

Full text of DOI:10.1021/ja981193h

J. Am. Chem. Soc. 1999, 121, 587-588
587
Communications to the Editor
Scheme 1
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
Scheme 2
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 R2â2 tetramer (R ) 59 000
Da, â ) 55 000 Da) and requires Fe(II), R-ketoglutarate, oxygen,
and ascorbate for activity.1 The genes for the R and â 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 [FeIV ) O]
intermediate would give radical 7. Recombination followed by
product dissociation would give the hemiacetal 9. In mechanism
B, â-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 transfer4 and product dissociation would
give 13 in which the oxaproline moiety has been oxidized to
aspartic acid.
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 (1H 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
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
(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.
(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,
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(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,
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10.1021/ja981193h CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/06/1999

588 J. Am. Chem. Soc., Vol. 121, No. 3, 1999
Communications to the Editor
Scheme 3
abstraction by a ferryl [FeIV ) O] intermediate to give a substrate
radical. In the systems for which radical probes have been
synthesized, experimental evidence in support of such a radical
intermediate has been difficult to obtain. The only successful
trapping experiment has been described using a cyclopropyl-
substituted cephalosporin analogue to trap the radical formed by
deacetoxy-deacetylcephalosporin C synthase.20 The inactivation
of γ-butyrobetaine hydroxylase by a cyclopropyl-containing
substrate analogue has been reported, but the reaction products
have not been characterized.21 A cyclopropyl-substituted procla-
vaminic acid analogue was not a substrate for clavaminate
synthase, and proline derivatives substituted with radical traps
were not substrates for prolyl-4-hydroxylase.22,23 The experiments
described here demonstrate a new strategy for radical-trapping
and suggest that the prolyl-4-hydroxylase-catalyzed oxidation of
proline residues in procollagen proceeds via a radical intermediate.
Studies to identify the labeled active-site residues of the
inactivated enzyme are currently in progress and will clarify how
the chemistry involved in the oxidation of 5 results in enzyme
inactivation.
Acknowledgment. We thank Mitch Brenner and Ari Koskinen for
helpful discussions and Cathy Lester for assistance in obtaining the 2D
NMR spectra. We are grateful to FibroGen, to the Academy of Finland
(Research Council for Health), and to the American Heart Association
for funding this research.
Supporting Information Available: Reaction schemes for the
synthesis of peptides 9 and 13 (2 pages, print/PDF). See any current
masthead page for ordering information and Web access instructions.
JA981193H
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The reaction product was then extracted into ethyl acetate and purified by
HPLC (ODS, 65:35 MeOH/H2O).
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