Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase

Abstract

The cofactor product of the aromatic amino acid hydroxylases, 4a-hydroxy-6(R)-tetrahydrobiopterin, requires dehydration before tetrahydrobiopterin can be regenerated by dihydropteridine reductase. Carbinolamine dehydration occurs nonenzymatically, but the reaction is also catalyzed by 4a-hydroxytetrahydropterin dehydratase. This enzyme has the identical amino acid sequence to DCoH, the dimerization cofactor of the transcription regulator, HNF-1 alpha. The catalytic activity of rat liver dehydratase was characterized using a new assay employing chemically synthesized 4a-hydroxytetrahydropterins. The enzyme shows little sensitivity to the structure or configuration of the 6-substituent of its substrate, with Km's for 6(S)-methyl, 6(R)-methyl, 6(S)-propyl, and 6(R)-L-erythro-dihydroxypropyl all between 1.5 and 6 microM. Turnover numbers at 37 degrees C are 50-90 s-1 at pH 7.4 and 2.5-3-fold lower at pH 8.4. Both 4a(R)- and 4a(S)-hydroxytetrahydropterins are good substrates. The quinoid dihydropterin products are strong inhibitors of the dehydratase with KI's about one half of their respective Km's, but no inhibition was observed with 7,8-dihydropterins or tetrahydropterins. The enzyme contains no metals and no phosphorus. Reaction mechanisms which involve either acid and/or base catalysis are discussed. 4a-Hydroxy-6(R)-tetrahydrobiopterin was determined not to be a product inhibitor of phenylalanine hydroxylase. It is concluded that the dehydratase (which was found to be 6 microM in rat liver) is essential in vivo to prevent rearrangement of 4a-hydroxy-6(R)-tetrahydrobiopterin and to maintain the supply of tetrahydrobiopterin cofactor for the hydroxylases under conditions where the nonenzymatic rate would be inadequate.

Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

“…A Hill coefficient of 1.8 and the sigmoidal curve shape indicate cooperativity between subunits of the tetrameric enzyme. This is in contrast to the reported characterization of PHS/DCoH with a synthetically generated substrate, where normal MichaelisMenten kinetics had been observed (27). However, a conformational change in the enzyme upon binding of pterins had been reported by Rebrin et al (28) and Ficner et al (29).…”

“…Rebrin et al (27). A Km of 3.0,uM and a Vmax of 16.5 ,tmol/min per mg (3.3 sec-1) have been reported with chemically synthesized 4a-OH-BH4 at pH 8.4 and 10°C (compare Table 2 (29) report that the solvent-accessible side chain of Cys-82 is not part of the pterin binding site but is in its vicinity.…”

Phylogenomic and Functional Analysis of Pterin-4a-Carbinolamine Dehydratase Family (COG2154) Proteins in Plants and Microorganisms

“…We used a functional complementation assay in Escherichia coli (Song et al, 1999;Wang et al, 2006) rather than in vitro assays because the latter involve artificial pterin4a-carbinolamine substrates (which may not necessarily be attacked by all PCDs) or lack sensitivity due to a background rate of chemical dehydration (Citron et al, 1992;Rebrin et al, 1995).…”

“…6A) and has some chemical characteristics of tetrahydropterins as well as others due to the third ring (Enemark and Garner, 1997;Nieter Burgmayer et al, 2004). Both substrate specificity studies (Rebrin et al, 1995) and the crystal structure of PCD complexed with a substrate analog (Cronk et al, 1996) indicate that PCD recognizes the pterin ring rather than its side chains. Thus, if Moco or its precursors form 4a-carbinolamines, they could be PCD substrates.…”

Structure and function of PCD/DCoH, an enzyme with regulatory properties

“…In contrast to the biotin repressor/synthase case, however, the connection between the enzymatic activity of pterin-4a-carbinolamine dehydratase (PCD) and its regulatory function as dimerisation cofactor of HNF1 (DCoH) is not well understood. PCD is involved in the regeneration of tetrahydrobiopterin (BH4), an essential cofactor of phenylalanine hydroxylase (PAH) and other mono-oxygenases, and catalyses the conversion of 4a-hydroxytetrahydrobiopterin to quinoid-dihydrobiopterin [4][5][6][7][8]. Certain human diseases like a mild form of hyperphenylalaninemia [9] and the depigmentation disorder vitiligo [10] are linked to a lack or deficiency of PCD activity.…”