Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata.
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 6.6. 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 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Krungkrai
J
J
Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021.
Cerami
A
A
Henderson
G B
GB
D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002850:Chromatography, Gel; D003421:Crithidia / Q000201:enzymology*; D004080:Dihydroorotase / Q000302:isolation & purification* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010962:Plasmodium berghei / Q000201:enzymology*; D011743:Pyrimidines / Q000096:biosynthesis*
D011743:Pyrimidines; C004768:4,5-dihydroorotic acid; D001224:Aspartic Acid; D009963:Orotic Acid; D004080:Dihydroorotase; C006893:ureidosuccinic acid; D004121:Dimethyl Sulfoxide