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Evidence for two discrete carbamyl phosphate pools in Neurospora.

L. Williams,S. Bernhardt,R. Davis

Published 1971 in Journal of Biological Chemistry

ABSTRACT

Abstract In Neurospora, carbamyl phosphate (carbamyl-P) is synthesized by two glutamine-dependent carbamyl phosphate synthetases (EC 2.7.2.5): one specific for the arginine pathway (CP-synthetase A), and one specific for the pyrimidine pathway (CP-synthetase P). Mutants lacking CP-synthetase A require arginine, and those lacking CP-synthetase P require uridine. Experiments were designed to test for the existence of two pathway-specific carbamyl-P pools. A method for determining carbamyl-P pools in exponentially growing Neurospora mycelia was developed. Steady state values of about 6 nmoles of carbamyl-P per g, dry weight, were obtained from wild type mycelium grown in minimal medium. Mutants having only CP-synthetase P contained 0.4 nmole of carbamyl-P per g, and mutants having only CP-synthetase A contained about 3.5 nmoles of carbamyl-P per g. Double mutants lacking both CP-synthetases contained no detectable carbamyl-P. A strain starving for uridine as a result of a CP-synthetase P deficiency has a normal or somewhat elevated pool of carbamyl-P produced by CP-synthetase A. The results indicate a "channeling" mechanism which normally segregates carbamyl-P of one pathway from the transcarbamylase of the other pathway. Impairment of transcarbamylase function, however, leads to a 10-fold elevation of the carbamyl-P associated with the pathway affected, and an overflow of carbamyl-P to the other pathway. The channeling mechanism, therefore, can be flooded by high carbamyl-P concentrations. The carbamyl-P associated with the pyrimidine sequence has a lower cellular concentration than the bifunctional enzyme complex carrying both CP-synthetase P and aspartate transcarbamylase activities. The complex could therefore confine pyrimidine-specific carbamyl-P as an enzyme-bound intermediate. Arginine-specific carbamyl-P may be confined in the mitochondrion, since a substantial fraction of CP-synthetase A and ornithine transcarbamylase is associated with this organelle in crude extracts. Regulation of the rate of arginine-specific carbamyl-P synthesis by arginine appears to take place mainly at the level of enzyme synthesis.

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