Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.

Abstract

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of a-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate a-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and or study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(Lproline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of a-ketoglutarate without itself being hydroxylated. All of these findings prove that a-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate. Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) catalyzes the hydroxylation of peptidyl proline with the concomitant decarboxylation of a-ketoglutarate. For each mole of proline hydroxylated, one mole of a-ketoglutarate is stoichiometrically decarboxylated to succinate and carbon dioxide. The enzyme also has an absolute requirement for ferrous ion and a reducing agent (1).The hydroxyl group of the hydroxyproline is derived from molecular. oxygen (2, 3). After the specific and absolute requirement of the enzyme for a-ketoglutarate was discovered (1, 4), several other oxygenase systems that require a-ketoglutarate were described (5-9). Lindblad et al. (10) reported that 180 from molecular oxygen is incorporated not only into carnitine during the y-butyrobetaine hydroxylase reaction, but also into the succinate derived from a-ketoglutarate. Cardinale et al. (11) were able to demonstrate that prolyl hydroxylase catalyzes the incorporation of equal amounts of 180 into peptidyl hydroxyproline and into the succinate derived from aketoglutarate. On the basis of such findings, Holme et al. (12) proposed as a mechanism for the hydroxylation of y-butyrobetaine that a peroxide bridge is formed between the a-ketoglutarate and y-butyrobetaine. Such an intermediate could then dissociate to yield the hydroxylated 'y-butyrobetaine (carnitine) and succinate. It was proposed (12) that a hydroperoxide is first formed on y-butyrobetaine; this hydroperoxide could then attack a-ketoglutarate and the intermediate would then decompose to carnitine, succinate, and CO2. An analogous reaction mechanism, involving the initial formation of a hydroperoxide on the number four carbon of the prolyl residue, can be postulated for prolyl hydro...

Prolyl 4-hydroxylase activity-responsive transcription factors: From hydroxylation to gene expression and neuroprotection

“…These studies reveal that the enzyme, isolated as a homogeneous protein by affinity chromatography from three different sources (8)(9)(10)12,14,15), occurs as a tetramer with a molecular weight of about 240,000 (11,(16)(17)(18). The enzyme does not hydroxylate free proline, and recognizes a conserved motif (LXXLAP in the HIF-1 molecule; X indicates any amino acid and P indicates the hydroxyl acceptor proline) in the primary substrate for hydroxylation (5,11,(19)(20)(21)(22). The hydroxylation of prolyl residues in this sequence is influenced by the nature of the amino acid in the X position, the nature of the amino acids in the adjacent sequences, the chain length and conformation of the primary substrate (11,16,23).…”

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

“…The decarboxylation step can proceed as an uncoupled reaction, i.e. without subsequent hydroxylation (Tuderman et al, 1977;Rao & Adams, 1978;Counts et al, 1978). Ascorbate serves as a specific alternative oxygen acceptor in such cycles (Myllyla et al, 1984;De Jong & Kemp, 1984), and in its absence the enzyme is rapidly inactivated by self-oxidation (Myllyli et al, 1978;De Jong et al, 1982).…”

Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

“…In PHD2, the dominant prolyl hydroxylase for HIF-1␣ protein in vivo, the Fe 2ϩ resides in a largely hydrophobic active site (42). During a complete reaction, the Fe 2ϩ is transiently oxidized to Fe 4ϩ and restored to the Fe 2ϩ state (13,46). However, when ␣-ketoglutarate is converted into succinate without hydroxylation of a peptide substrate, this Fe 2ϩ is oxidized to Fe 3ϩ during the reaction.…”

“…However, when ␣-ketoglutarate is converted into succinate without hydroxylation of a peptide substrate, this Fe 2ϩ is oxidized to Fe 3ϩ during the reaction. During this process ascorbate is required to re-duce the Fe 3ϩ back to Fe 2ϩ in order for the enzyme to be recycled (13,46). Therefore, intracellular ascorbate and Fe 2ϩ levels can also affect PHD enzymatic activity and HIF-␣ protein accumulation (33,34).…”