CATALYTIC CHARACTERIZATION OF 4A-HYDROXYTETRAHYDROPTERIN DEHYDRATASE

The cofactor product of the aromatic amino acid hydroxylases, 4a-hydro xy-6(R)-tetrahydrobiopterin, requires dehydration before tetrahydrobio pterin can be regenerated by dihydropteridine reductase. Carbinolamine dehydration occurs nonenzymatically, but the reaction is also catalyz ed by 4a-hydroxytetrahydropterin dehydratase. This enzyme has the iden tical amino acid sequence to DCoH, the dimerization cofactor of the tr anscription regulator, HNF-1 alpha. The catalytic activity of rat five r dehydratase was characterized using a new assay employing chemically synthesized 4a-hydroxytetrahydropterins. The enzyme shows little sens itivity to the structure or configuration of the 6-substituent of its substrate, with K-m's for 6(S)-methyl, 6(R)-methyl, 6(S)-propyl, and 6 (R)-L-erythro-dihydroxypropyl all between 1.5 and 6 mu M. Turnover num bers 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 subst rates. The quinoid dihydropterin products are strong inhibitors of the dehydratase with K-I's about one half of their respective K-m's, but no inhibition was observed with 7,8-dihydropterins or tetrahydropterin s, The enzyme contains no metals and no phosphorus. Reaction mechanism s which involve either acid and/or base catalysis are discussed. 4a-Hy droxy-6(R)tetrahydrobiopterin was determined not to be a product inhib itor of phenylalanine hydroxylase. It is concluded that the dehydratas e (which was found to be 6 mu M in rat liver) is essential in vivo to prevent rearrangement of 4a-hydroxy-6(R)-tetrahydrabiopterin and to ma intain the supply of tetrahydrobiopterin cofactor for the hydroxylases under conditions where the nonenzymatic rate would be inadequate.