Dihydrooxonate is a substrate of dihydroorotate dehydrogenase (DHOD) providing evidence for involvement of cysteine and serine residues in base catalysis

The flavoprotein dihydroorotate dehydrogenase (DHOD) catalyzes the oxidatio n of dihydroorotate to orotate. Dihydrooxonate is an analogue of dihydrooro tate in which the C5 carbon is substituted by a nitrogen atom. We have inve stigated dihydrooxonate as a substrate of three DHODs, each representing a distinct evolutionary class of the enzyme, namely the two family 1 enzymes from Lactococcus lactis, DHODA and DHODB, and the enzyme from Escherichia c oli, which, like the human enzyme, belongs to family 2. Dihydrooxonate was accepted as a substrate although much less efficiently than dihydroorotate. The first half-re action was rate limiting according to pre-steady-state a nd steady-state kinetics with different electron accepters. Cysteine and se rine have been implicated as active site base residues, which promote subst rate oxidation in family 1 and family 2 DHODs, respectively. Mutants of DHO DA (C130A) and E. coli DHOD (S175A) have extremely low activity in standard assays with dihydroorotate as substrate, but with dihydrooxonate the mutan ts display considerable and increasing activity above pH 8.0. Thus, the abs ence of the active site base residue in the enzymes seems to be compensated for by a lower pK(alpha) of the 5-position in the substrate. Oxonate, the oxidation product of dihydrooxonate, was a competitive inhibitor versus dih ydroorotate, and DHODA was the most sensitive of the three enzymes. DHODA w as reinvestigated with respect to product inhibition by orotate. The result s suggest a classical one-site ping-pong mechanism with fumarate as electro n acceptor, while the kinetics with ferricyanide is highly dependent on the detailed reaction conditions. (C) 2001 Academic Press.