Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata

Abstract

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 .+-. 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 .+-. 3000. The DHOase from P. berghei had a native molecular weight of 40,000 .+-. 4000 and a subunit molecular weight on SDS-PAGE of 38,000 .+-. 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 7.7. The Km and kcat for L-CA were 0.846 .+-. 0.017 mM and 39.2 .+-. 6.4 min-1, respectively; for L-DHO, they were 25.85 .+-. 2.67 .mu.M and 258.6 .+-. 28.5 min-1. The cryoprotectant dimethyl sulfoxide (Me2SO), used as stabilizing agent in the complete purification and storage, markedly affected the DHOase activity. Me2SO increased the catalytic efficiency of the enzyme, as measured by kcat/Km, in the ring cyclization reaction but had no effect on the ring cleavage reaction. In spite of their marked physical differences, these kinetic and inhibitor studies with 5-substituted derivatives of orotic acid suggest that the protozoan, mammalian, and prokaryotic enzymes have a common catalytic mechanism.