The mechanism for the reaction is similar to that of other dioxygenases, and occurs in two distinct stages[4]: In the first, a highly reactive
Fe(IV)=O species is produced. Molecular oxygen is bound end-on in an axial position, producing a dioxygen unit. Nucleophilic attack on C2 generates a tetrahedral intermediate, with loss of the double bond in the dioxygen unit and bonds to iron and the alpha carbon of 2-oxoglutarate. Subsequent elimination of CO2 coincides with the formation of the Fe(IV)=O species. The second stage involves the abstraction of the pro-R hydrogen atom from C-4 of the proline substrate followed by radical combination, which yields hydroxyproline[5].
As a consequence of the reaction mechanism, one molecule of 2-oxoglutarate is
decarboxylated, forming succinate. This succinate is
hydrolyzed and replaced with another 2-oxoglutarate after each reaction, and it has been concluded that in the presence of 2-oxoglutarate, enzyme-bound Fe2+ is rapidly converted to Fe3+, leading to inactivation of the enzyme[6]. Ascorbate is utilized as a
cofactor to reduce Fe3+ back to Fe2+[7].
Enzyme Structure
Prolyl hydroxylase is a
tetramer with 2 unique subunits[8]. The α subunit is 59 kDa and is responsible for both peptide binding and for catalytic activity[9]. The peptide binding domain spans residues 140-215 of the α subunit[10], and consists of a concave surface lined with multiple
tyrosine residues which interact favorably with the proline-rich substrate. The
active site consists of Fe2+ bound to two
histidine residues and one
aspartate residue, a characteristic shared by most 2-oxoglutarate-dependent dioxygenases. The 55 kDa β subunit is responsible for the enzyme’s localization to and
retention in the endoplasmic reticulum.[11] Interestingly, this subunit is identical to the enzyme known as
protein disulfide isomerase[12].
Biological Function
Prolyl hydroxylase catalyzes the formation of hydroxylation of proline, which is the most abundant post-translational modification in human body. The modification has a significant impact on the stability of collagen, the major connective tissue of the human body[13]. Specifically, hydroxylation increases the melting temperature of helical collagen by 16°C, as compared to unhydroxylated collagen [14], a difference that allows the protein to be stable at body temperature.
The enzyme acts specifically on proline contained within the X-Pro-Gly motif – where Pro is proline. Because of this motif-specific behavior, the enzyme also acts on other proteins that contain this same sequence. Such proteins include
C1q[15],
elastins[16],
PrP[17],
Argonaute 2[18], and
conotoxins[19], among others.
Disease Relevance
As prolyl hydroxylase requires ascorbate as a cofactor to function[20], its absence compromises the enzyme’s activity. The resulting decreased hydroxylation leads to the disease condition known as
scurvy. Since stability of collagen is compromised in scurvy patients, symptoms include weakening of blood vessels causing
purpura,
petechiae, and gingival bleeding.
Alternate Names
Protocollagen hydroxylase
Prolyl hydroxylase
Prolyl 4-hydroxylase
Protocollagen prolyl hydroxylase
^Smith, T. G.; Talbot, N. P. (2010). "Prolyl hydroxylases and therapeutics". Antioxidants & Redox Signaling. 12 (4): 431–3.
doi:
10.1089/ars.2009.2901.
PMID19761407.
^Hutton Jr, J. J.; Trappel, A. L.; Udenfriend, S (1966). "Requirements for alpha-ketoglutarate, ferrous ion and ascorbate by collagen proline hydroxylase". Biochemical and Biophysical Research Communications. 24 (2): 179–84.
doi:
10.1016/0006-291x(66)90716-9.
PMID5965224.
^Fujita, Y.; Gottlieb, A.; Peterkofsky, B.; Udenfriend, S.; Witkop, B. (1964). "The Preparation of cis- and trans-4-H3-L-Prolines and Their Use in Studying the Mechanism of Enzymatic Hydroxylation in Chick Embryos". Journal of the American Chemical Society. 86 (21): 4709.
doi:
10.1021/ja01075a036.
^De Jong, L; Albracht, S. P.; Kemp, A (1982). "Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation". Biochimica et Biophysica Acta. 704 (2): 326–32.
doi:
10.1016/0167-4838(82)90162-5.
PMID6285984.
^De Jong, L; Kemp, A (1984). "Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction". Biochimica et Biophysica Acta. 787 (1): 105–11.
doi:
10.1016/0167-4838(84)90113-4.
PMID6326839.
^Berg, R. A.; Prockop, D. J. (1973). "Affinity column purification of protocollagen proline hydroxylase from chick embryos and further characterization of the enzyme". The Journal of biological chemistry. 248 (4): 1175–82.
PMID4346946.
^Pekkala, M; Hieta, R; Bergmann, U; Kivirikko, K. I.; Wierenga, R. K.; Myllyharju, J (2004). "The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a tetratricopeptide repeat domain with functional aromatic residues". Journal of Biological Chemistry. 279 (50): 52255–61.
doi:
10.1074/jbc.M410007200.
PMID15456751.{{
cite journal}}: CS1 maint: unflagged free DOI (
link)
^Berg, R. A.; Prockop, D. J. (1973). "The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen". Biochemical and Biophysical Research Communications. 52 (1): 115–20.
doi:
10.1016/0006-291x(73)90961-3.
PMID4712181.
^Müller, W; Hanauske-Abel, H; Loos, M (1978). "Biosynthesis of the first component of complement by human and guinea pig peritoneal macrophages: Evidence for an independent production of the C1 subunits". Journal of immunology (Baltimore, Md. : 1950). 121 (4): 1578–84.
PMID701808.
^Rosenbloom, J; Cywinski, A (1976). "Inhibition of proline hydroxylation does not inhibit secretion of tropoelastin by chick aorta cells". FEBS Letters. 65 (2): 246–50.
doi:
10.1016/0014-5793(76)80490-5.
PMID6335.
^Daly, N. L.; Craik, D. J. (2009). "Structural studies of conotoxins". IUBMB Life. 61 (2): 144–50.
doi:
10.1002/iub.158.
PMID19165896.
^De Jong, L; Albracht, S. P.; Kemp, A (1982). "Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation". Biochimica et Biophysica Acta. 704 (2): 326–32.
doi:
10.1016/0167-4838(82)90162-5.
PMID6285984.