Dihydrooxonate Is a Substrate of Dihydroorotate Dehydrogenase (DHOD) Providing Evidence for Involvement of Cysteine and Serine Residues in Base Catalysis

摘要: The flavoprotein dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of to orotate. Dihydrooxonate is an analogue in which C5 carbon substituted by a nitrogen atom. We have investigated dihydrooxonate as substrate three DHODs, each representing distinct evolutionary class enzyme, namely two family 1 enzymes from Lactococcus lactis, DHODA and DHODB, enzyme Escherichia coli, which, like human belongs 2. was accepted although much less efficiently than dihydroorotate. first half-reaction rate limiting according pre-steady-state steady-state kinetics with different electron acceptors. Cysteine serine been implicated active site base residues, promote 2 respectively. Mutants (C130A) E. coli DHOD (S175A) extremely low activity standard assays substrate, but mutants display considerable increasing above pH 8.0. Thus, absence residue seems be compensated for lower pK(a) 5-position substrate. Oxonate, product dihydrooxonate, competitive inhibitor versus dihydroorotate, most sensitive enzymes. reinvestigated respect inhibition results suggest classical one-site ping-pong mechanism fumarate acceptor, while ferricyanide highly dependent on detailed reaction conditions.