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 eect
was determined to be 3.4 Æ 0.2. This isotope eect 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 eect 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 eect on the reaction when procollagen
was used as the substrate.7 We anticipated that it might
be possible to detect such an isotope eect 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 eect
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 eect 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 eect 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 eect was detected on V/K.
In general, a V/K isotope eect 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
Wu, Min
