Mechanistic studies on prolyl-4-hydroxylase: The vitamin C requiring uncoupled oxidation

Article abstract of DOI:10.1016/S0960-894X(00)00224-9

A deuteriated substrate for the human type I prolyl-4-hydroxylase was synthesized and its V/K deuterium isotope effect was determined to be 3.4 ± 0.2. The isotope effect was attributed to the uncoupled oxidation. A dehydroproline containing tetrapeptide was also found to stimulate the uncoupled oxidation. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00224-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1511±1514
Mechanistic Studies on Prolyl-4-hydroxylase: The Vitamin C
Requiring Uncoupled Oxidation
Min Wu,a Hong-sik Moon,a Asta Pirskanen,b Johanna Myllyharju,b
Kari I. Kivirikkob and Tadhg P. Begleya,*
aDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
bCollagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu,
Kajaanintie 52A, FIN-90220, Oulu, Finland
Received 20 December 1999; accepted 10 April 2000
AbstractÐA deuteriated substrate for the human type I prolyl-4-hydroxylase was synthesized and its V/K deuterium isotope e€ect
was determined to be 3.4 Æ 0.2. This isotope e€ect was attributed to the uncoupled oxidation. A dehydroproline containing tetra-
peptide was also found to stimulate the uncoupled oxidation. # 2000 Elsevier Science Ltd. All rights reserved.
Prolyl-4-hydroxylase catalyzes the hydroxylation of
prolyl residues at X-Pro-Gly sequences in procollagen
(Scheme 1). This reaction is an essential step in the bio-
synthesis of collagen, the major protein component of
connective tissue.1,2
To determine the kinetic signi®cance of the C±H bond
cleavage step in the overall reaction Scheme, we have
measured the V/K deuterium isotope e€ect on the reac-
tion using N-Cbz-Gly-Phe-Pro-Gly-OEt as the substrate
20.6 Previous research demonstrated that there was no
V/K isotope e€ect on the reaction when procollagen
was used as the substrate.7 We anticipated that it might
be possible to detect such an isotope e€ect with the
smaller substrate because of the smaller forward com-
mitment to catalysis for 20 compared to procollagen.
The current mechanistic proposal for this enzyme is out-
lined in Scheme 2.3 5 and involves the hydroxylation of
the substrate by the ferryl intermediate 7. In addition
to this productive reaction, a nonproductive oxidation
resulting in enzyme inactivation occurs.2 The mechanism
of this uncoupled oxidation is not understood. One
possibility is that the ferryl intermediate 7 oxidizes the
enzyme to an inactive form 8, which requires reduction
by ascorbate for reactivation. The uncoupled oxidation
is likely to be one of the most important ascorbate
requiring enzymatic reactions in humans because the
earliest clinical symptoms of scurvy, the vitamin C de®-
ciency disease, are defects in collagen biosynthesis. The
function of this inactivation reaction may be to protect
the enzyme from a more destructive irreversible oxida-
tion event. Here we describe a deuterium isotope e€ect
and the interaction of a dehydroproline-containing
substrate analogue with the enzyme as two possible
mechanistic probes for this reaction.
The synthesis of the deuteriated substrate 19 is outlined
in Scheme 3. The nondeuteriated substrate 20 was syn-
thesized in an identical manner from N-Boc-Pro. The V/
K isotope e€ect was determined by incubating a mixture
of 19 and 20 with the human enzyme,8 extracting the
reaction mixture with dichloromethane and measuring
the deuterium content of the extracted substrate and
product by FABMS. This mode of analysis was possible
because of the deuterium label on the ester of 19. The V/
K isotope e€ect was then calculated as described by
Cleland9 and found to be 3.4 Æ 0.2 (four determinations).
When the reaction rate was determined by measuring
the rate of carbon dioxide production from a-keto-
glutarate, no isotope e€ect was detected on V/K.
In general, a V/K isotope e€ect will be observed if the
C±H bond cleavage is kinetically signi®cant and occurs
at or before the ®rst irreversible step in the reaction
sequence. Since the formation of carbon dioxide and the
*Corresponding author. Tel.: +1-607-255-7133; fax: +1-607-255-
4137; e-mail: tpb2@cornell.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00224-9

1512
M. Wu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1511±1514
Scheme 1.
Scheme 2.
ferryl intermediate 7 is very unlikely to be reversible, it
is not surprising that there is no V/K isotope e€ect on
the rate of carbon dioxide formation. The observed V/K
isotope e€ect on the rate of the substrate hydroxylation
is likely to result from the uncoupled oxidation which
makes it possible for 7 to return to free enzyme and
peptide substrate. Thus, with the deuterio substrate, a
primary isotope e€ect on the hydrogen atom abstraction
would result in a greater amount of enzyme oxidation to
give 8 than would occur with the protio substrate.10 12
We therefore propose that the V/K isotope e€ect on
prolyl-4-hydroxylase may be a useful mechanistic probe
for studying the uncoupled oxidation.
proline-3-hydroxylase and proline-4-hydroxylase catalyze
the epoxidation of dehydroproline.15,16 We tested this
hypothesis using the small dehydroproline containing pep-
tide 22, which was synthesized as outlined in Scheme 4.17
When 22 was incubated with prolyl-4-hydroxylase
under standard assay conditions,8 no product formation
was detected by chromatographic and MS analysis of
the dichloromethane extracted peptide. However, when
the enzymatic reaction was monitored by measuring the
rate of carbon dioxide formation from a-ketoglutarate,
22 appeared to be a very good substrate for the enzyme
(Km=200 mM, Vmax=95%, compared to Km=520 mM,
Vmax=30% for N-Cbz-Gly-Phe-Pro-Gly-OEt and
Km=200 mM and Vmax=100% for (Pro-Pro-Gly)10).
This suggests that the uncoupled oxidation is the sole
reaction pathway for the dehydroproline containing
peptide and that 22 may also be a useful mechanistic
probe for this reaction.18 20
The inhibition of prolyl-4-hydroxylase by dehydropro-
line has been previously described.13,14 A reasonable
mechanism for this inhibition involves the epoxidation of
the double bond followed by alkylation of the enzyme.
This proposal is supported by the observation that both

M. Wu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1511±1514
1513
Scheme 3.
Scheme 4.
4. Wu, M.; Moon, H.-S.; Begley, T. P.; Myllyharju, J.; Kivir-
ikko, K. I. J. Am. Chem. Soc. 1999, 121, 587.
Acknowledgements
5. Que, L.; Ho, R. Y. Chem. Rev. 1996, 96, 2607.
6. Tandon, M.; Wu, M.; Begley, T. P.; Myllyharju, J.; Pirska-
nen, A.; Kivirikko, K. I. Bioorg. Med. Chem. Lett. 1998, 8,
1139.
7. Ebert, P. S.; Prockop, D. J. Biochem. Biophys. Res. Comm.
1962, 8, 305.
Support for this research by grants from the American
Heart Association and from FibroGen Inc. is gratefully
acknowledged.
8. A typical enzymatic reaction mixture consisted of: peptide
(0.85 mM), FeSO4 (0.05 mM), ascorbic acid (2.0 mM), BSA
(1 mg), catalase (0.05 mg), DTT (0.1 mM), a-ketoglutarate
(0.5 mM) Tris±HCl (50 mM, pH 7.8) prolyl-4-hydroxylase
(68 mg) in 500 mL. Vuori, K.; Pihlajaniemi, T.; Marttila, M.;
Kivirikko, K. I. Proc. Natl. Acad. Sci. USA 1992, 89, 7467.
9. Cleland, W. W. Crit Rev. Biochem. 1982, 13, 385.
10. V/K isotope e€ects due to the uncoupled oxidation have
previously been reported for g-butyrobetaine hydroxylase and
for clavaminate synthase.11,12
References and Notes
1. Kivirikko, K. I.; Pihlajaniemi, T. Adv. Enzymol. Related
Areas Mol. Biol. 1998, 72, 325.
2. Kivirikko, K. I.; Myllyla, R.; Pihlajaniemi, T. In Post
Translational Modi®cation of Proteins; Harding, J.; Crabbe,
M., Eds.; CRC: 1992; pp 1±51.
3. Hanauske-Abel, H. M.; Gunzler, V. J. Theor. Biol. 1982, 94,
421.

1514
M. Wu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1511±1514
11. Salowe, S. P.; Krol, W. J.; Iwata-Reuyl, K.; Townsend, C.
A. Biochemistry 1991, 30, 2281.
12. Holme, E.; Lindstedt, S.; Nordin, I. Bioscience Reports
1984, 4, 433.
13. Nolan, J. C.; Ridge, S.; Oronsky, A. L.; Kerwar, S. S.
Arch. Biochem. Biophys. 1978, 189, 448.
14. Kerwar, S. S.; Marcel, R. J. M.; Salvador, R. A. Arch.
Biochem. Biophys. 1976, 172, 685.
Mori, H.; Yoshida, M.; Ozaki, A. Tetrahedron Lett. 1999, 40,
5227.
17. All new compounds were characterized by 1H NMR.
Compounds 19 and 20 were also characterized by HR±MS.
18. Enhancement of the uncoupled oxidation using substrate
analogues has been previously reported for thymine hydro-
xylase and g-butyrobetaine hydroxylase.19,20
19. Thornburg, L. D.; Lai, M. T.; Wishnok, J. S.; Stubbe, J.
Biochemistry 1993, 32, 14023.
15. Baldwin, J.; Field, R.; Lawrence, C.; Lee, V.; Robinson, J.;
Scho®eld, C. Tetrahedron Lett. 1994, 35, 4649.
16. Shibasaki, T.; Sakurai, W.; Hasegawa, A.; Uosaki, Y.;
20. Wehbie, R. S.; Punekar, N. S.; Lardy, H. A. Biochemistry
1988, 27, 2222.