The catalytic mechanism of hamster dihydroorotase.

Abstract

Dihydroorotase catalyses the third reaction of the pathway for de novo biosynthesis of pyrimidine nucleotides. The reaction catalysed involves an intramolecular ring closure of N-carbamyl-L-aspartate (CA-asp) to form L-dihydroorotate (DHO), a dihydropyrimidine. The interconversion of CA-asp and DHO is freely reversible (Christopherson and Jones, 1979) and the pH-rate profiles for the biosynthetic and degradative reactions and inhibition of the enzyme by L-cysteine suggested that mammalian dihydroorotase has a catalytic mechanism similar to carboxypeptidase A (Christopherson and Jones, 1980) with a zinc atom at the active site. This zinc atom would polarise the =C=O group of the scissile peptide bond in the dihydropyrimidine ring of DHO and stabilise the tetrahedral, dioxy anionic transition state of the reaction (Fig. 1). Taylor et al. (1976) had purified the dihydroorotase from Clostridium oroticum and found two gram atoms of zinc per subunit and similar pH-rate profiles. Kelly et al. (1986) subsequently demonstrated that hamster dihydroorotase also contained an atom of zinc which was required for catalysis. It therefore seems likely that mammalian dihydroorotase does have a catalytic mechanism which resembles that of zinc protease.