Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).