Prolyl 4-hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets

Figures & data

Table

C-P4H	collagen prolyl 4-hydroxylase
ER	endoplasmic reticulum
Epo	erythropoietin
HIF	hypoxia-inducible transcription factor
PDI	protein disulfide isomerase
ODDD	oxygen-dependent degradation domain
SiRNA	short interfering RNA
P4H-TM	transmembrane P4H
TCA	tricarboxylic acid cycle
VEGF	vascular endothelial growth factor
VHL	von Hippel-Lindau

Figure 1.  Hydroxylation of proline residues by C-P4Hs is essential for the thermal stability of collagen triple helices. Non-hydroxylated collagen polypeptide chains cannot form functional molecules in vivo.

Figure 2.  Regulation of the stability of the HIF-1α by oxygen-dependent prolyl 4-hydroxylation. Under normoxic conditions, HIF-1α is hydroxylated by HIF-P4Hs. Hydroxylation is required for binding of the VHL E3 ubiquitin ligase complex and for subsequent proteasomal degradation. Hypoxia inhibits the HIF-P4Hs, HIF-1α escapes degradation and forms a stable dimer with HIF-β. The dimer is translocated into the nucleus and binds to the HIF-responsive elements in a number of hypoxia-inducible genes.

Figure 3.  Schematic representation of the human C-P4H α(I), α(II) and α(III) subunits, P4H-TM and HIF-P4Hs 1–3. The lengths of the polypeptides are indicated on the right, and the catalytically critical residues are shown above the polypeptides. The peptide-substrate- binding domain in the C-P4H α subunits and the TM domain of P4H-TM are also indicated.

Figure 4.  Reactions catalyzed by P4Hs. 2-Oxoglutarate is stoichiometrically decarboxylated during the hydroxylation reaction, which does not require ascorbate (A). The enzymes also catalyze the uncoupled decarboxylation of 2-oxoglutarate without subsequent hydroxylation of the peptide substrate. Ascorbate is stoichiometrically consumed in the uncoupled reaction, which may occur in either the presence (B) or absence (C) of the peptide substrate.]

Figure 5.  Schematic representation of the catalytic site of the α(I) subunit of human C-P4H-I. Fe^2 + is coordinated with the enzyme by His412, Asp414, and His483. Subsite I of the 2-oxoglutarate binding site consists of Lys493, which ionically binds the C5 carboxyl group of 2-oxoglutarate, while subsite II consists of two cis-positioned equatorial coordination sites of the enzyme-bound Fe^2 + and is chelated by the C1 carboxyl and C2 oxo functions of the 2-oxoglutarate. Molecular oxygen is bound end-on in an axial position, producing a dioxygen unit.

Table I.  K_m values of recombinant human C-P4Hs I-III and HIF-P4Hs 1-3 for Fe^2 +, 2-oxoglutarate, O₂ and ascorbate^a.

Cosubstrate	C-P4H-I	C-P4H-II	C-P4H-III	HIF-P4H-1	HIF-P4H-2	HIF-P4H-3
	Km, µM
Fe^2 +	2	2	0.5	0.03	0.03	0.1
2-Oxoglutarate	20	22	20	2	1	12
O2	40	N.D.^b	N.D.	230	250^c	230
O2					100^d
O2					65–85^e
Ascorbate	300	340	370	170	180	140

^aRefs. 8, 9, 50, 54, 61, 87-90.^bNot determined.^cK_m for O₂ determined with a 19-residue HIF1α peptide.^dK_m for O₂ determined with a 248-residue HIF1α ODDD fragment.^eK_m for O₂ determined with 123-195-residue HIF1α and HIF2α ODDD fragments.

Table II.  Inhibition of purified recombinant C-P4H-I and HIF-P4Hs 1-3 by certain metals and 2-oxoglutarate analogues^a.

Inhibitor	C-P4H-I	HIF-P4H-1	HIF-P4H-2	HIF-P4H-3
	IC50, µM
Zn^2 +	0.6^b	28	130	4
Co^2 +	14	38	100	9
Ni^2 +	37	130	>1000	120
	Ki, µM
Pyridine 2,4-dicarboxylate	2	40	7	8
Pyridine 2,5-dicarboxylate	0.8	>300	>300	>300
3-Hydroxypyridine-2-carbonyl-glycine	0.4	15	2	1
Oxalylglycine	1.9	50	8	10
3,4-Dihydroxybenzoic acid	5	>300	>300	>300
3-Carboxy-4-oxo-3,4-dihydro-1,10-phenanthroline	2	30	10	10
N-((3-hydroxy-6-chloroquinolin-2-yl)carbonyl)glycine	0.06	0.8	0.2	0.2
Fumarate	190	80	60	50
Succinate	400	350	460	430

^aRefs. 9, 61, 87.