Urea carboxylase and allophanate hydrolase. Two components of adenosine triphosphate:urea amido-lyase in Saccharomyces cerevisiae.

The ATP:urea amido-lyase reaction of yeast has been shown. to consist of two distinct activities, an avidin-sensitive, thiourea-insensitive urea carboxylase and an avidin-insensitive, thiourea-sensitive allophanate hydrolase. Mutants lacking each of these activities have been isolated and were found to complement each other. Enzyme preparations and assay procedures were similar to those described previously (1). The specific activities of the preparations used in this work ranged from 0.2 to 0.6 and 1.2 to 1.6 units per mg for urea-dependent ADP production and allophanate-dependent COZ production, respectively. M-25, the wild type strain employed, is the prototrophic diploid resulting from the cross, XT1172S185 (a), ade, ler) x XT1172S62 (a, hie, ly,, un). A series of mutants unable to grow on urea as the sole nitrogen source were isolated following EMS mutagenesis (8) of XT1172S185. The mutagenized culture was diluted and plated to give approximately 100 colonies per plate on YEPD agar (9) containing 0.1% (NH&S04. After 2 days at 30°, each plate was replicated to a series of four minimal (10) plates containing 2% glucose, 120 pg per ml of leucine, 20 pg per ml of adenine, and a nitrogen source to screen for those colonies specifically deficient in urea metabolism. Nitrogen sources for the four types of plates were 1.0 X lo+ M urea, 0.1 $Z$ (NH&SO+ 3.7 X 10e3 M arginine, and 7.5 X low3 M ornithine, respectively. Purification of mutants, complementation tests, and construction of prototrophic diploids carrying the various mutant alleles were performed employing standard genetic methods (9). M-62 and M-64 are prototrophic diploids homozygous for mutant alleles E-145 and E-142, respectively. Growth of the mutants for determination of the enzymes was accomplished on minimal medium containing 0.6% glucose and 0.02% (NH&SO*. Urea, 1.0 x lo-* M, was added at a cell density of approximately 6 X lo6 cells per ml and after one generation of growth the cultures were made 10 pg per ml in cycloheximide and the cells were collected by centrifugation. After resuspension in 2.5 ml of 0.05 M Tris, pH 7.9, containing 57< glycerol, 2.0 X lop4 M EDTA, and 3.0 X low3 M mercaptoethanol, the cells were permeabilized with the method of Ramos et al. (ll), with the exception that the procedures were carried out at 0”. Saccharomyces cerevisiae grown on urea as a nitrogen source contains an avidin-sensitive urea degradation system which has been described (1, 2) by the reaction