A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme
The pyrimidine-3 locus of Neurospora crassa specifies a multienzyme complex comprising pyrimidine-specific carbamoyl phosphate synthase (CPSpyr) and aspartate carbamoyl transferase (ACT). It appears to be divided into a translationally proximal CPS-specific region and a distal ACT-specific region.
Levels of complementation for ACT activity between pairs of four pyr-3 CPS+ACT- mutants showed a range from 12% to 68% of the wild-type level of the enzyme. This is interpreted as interallelic complementation, contradicting certain earlier suggestions of two dissimilar ACT subunits.
Proteolysis of an extract from a heterokaryon formed from two of the above CPS+ACT- alleles (α and β) did not lead to loss of ACT activity, but led to the formation of a fragment with ACT activity with a similar molecular weight (92,000 daltons) to that produced in extracts of wild type strain.
The pyr-3 polar mutant 43–174, which is enzymatically CPS+ACT- and which fails to complement with any other CPS+ACT- alleles, thus suggesting its location towards the proximal end of the ACT region, has CPS activity associated with a form of 180,000 daltons molecular weight. These findings are used to construct a model for the structure of the native enzyme complex.
KeywordsEnzyme Wild Type Strain Native Enzyme Neurospora Crassa Carbamoyl
Unable to display preview. Download preview PDF.
- Abd-el-Al, A., Ingraham J.L.: Carbamyl phosphate synthetase from Salmonella typhimurium: Regulation, subunit composition, and function of subunits. J. biol. Chem. 250, 4410–4417 (1975)Google Scholar
- Aitken, D.M., Lue, P.F., Kaplan, J.G.: Characterization of the aspartate transcarbamylase subunit from a multi-enzyme aggregate in the pyrimidine pathway of yeast. Biochim. biophys. Acta (Amst.) 309, 50–57 (1973)Google Scholar
- Anderson, P.M., Marvin, S.V.: Effect of ornithine, IMP, and UTP on carbamyl phosphate synthetase from Escherichia coli Biochem. biophys. Res. Commun. 32, 928–934 (1968)Google Scholar
- Brabson, J.S., Switzer, R.L.: Purification and properties of Bacillus subtilis aspartate transcarbamylase. J. biol. Chem. 250, 8664–8669 (1975)Google Scholar
- Chang, T.Y., Jones, M.E.: Aspartate transcarbamylase from Streptococcus faecalis. Purification, properties, and nature of an allosteric activator site. Biochemistry 13, 629–638 (1974)Google Scholar
- Davis, R.H.: An enzymatic difference among pyr-3 mutants of Neurospora crassa. Proc. nat. Acad. Sci. (Wash.) 46, 667–682 (1960)Google Scholar
- Davis, R.H.: Channeling in Neurospora metabolism, pp. 303–322. In: Organisational biosynthesis (Vogel, H.J., Lampen, L.O. Bryson, V., eds.). New York: Academic Press Inc. 1967Google Scholar
- Denis-Duphil, M., Lacroute, F.: Fine structure of the ura2 locus in Saccharomyces cerevisiae. 1. In vivo complementation studies. Molec. gen. Genet. 112, 354–364 (1971)Google Scholar
- Gerhart, J.C., Schachman, H.K.: Distinct subunits for the regulation and catalytic activity of aspartate transcarbamylase. Biochemistry 4, 1054–1062 (1965)Google Scholar
- Issaly, I.M., Issaly, A.S., Reissig, J. L.: Carbamyl phosphate synthesis in Bacillus subtilis. Biochim. biophys. Acta (Amst.) 198, 482–494 (1970)Google Scholar
- Lowry, O.H., Rosebrough, N.J., Farr, A. L., Randall, J.R. Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)Google Scholar
- Lue, P.F., Kaplan, J.G.: Aggregation states of a regulatory enzyme complex catalyzing the early steps of pyrimidine biosynthesis in bakers' yeast. Canad. J. Biochem. 49, 403–411 (1971)Google Scholar
- Makoff, A.J.: Characterization of the aspartate carbamoyl transferase fragment generated by protease action of the pyrimidine-3 gene product of Neurospora crassa. Biochim. biophys. Acta (Amst.) 485, 314–329 (1977)Google Scholar
- Makoff, A.J., Radford, A.: The location of the feedback-specific region within the pyrimidine-3 locus of Neurospora crassa. Molec. gen. Genet. 146, 247–251 (1976)Google Scholar
- Makoff, A.J., Radford, A.: Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of Neurospora crassa. Molec. gen. Genet. 149, 175–178 (1976)Google Scholar
- Makoff, A.J., Radford, A.: Dissociation of an enzyme complex from Neurospora crassa: evidence for a protease. Biochim. biophys. Acta (Amst.) 485, 314–329 (1977)Google Scholar
- Mergeay, M., Gigot, D., Beckmann, J., Glansdorff, N., Pierard, A.: Physiology and genetics of carbamyl phosphate synthesis in Escherichia coli K12. Molec. gen. Genet. 133, 299–316 (1974)Google Scholar
- Palmer, L.M., Cove, D.J.: Pylimidine biosynthesis in Aspergillus nidulans. Isolation and preliminary characterization of auxotrophic mutants. Molec. gen. Genet. 138, 243–255 (1975)Google Scholar
- Prescott, L.M., Jones, M.E.: Modified methods for determination of carbamyl aspartate. Analyt. Biochem. 32, 408–419 (1969)Google Scholar
- Radford, A.: Information from ICR-170-induced mutations on the structure of the pyr-3 locus in Neurospora. Mutation Res. 8, 537–544 (1969)Google Scholar
- Radford, A.: Pyrimidine-requiring suppressor mutations of arg-3 in Neurospora and their bearing on the structure of the pyr-3 locus. Molec. gen. Genet. 107, 97–106 (1970a)Google Scholar
- Radford, A.: Intragenic mapping of the Neurospora pyr-3 locus by functional deletions. Molec. gen. Genet. 109, 241–245 (1970b)Google Scholar
- Reissig, J.L.: Spectrum of forward mutants in the pyr-3 region of Neurospora. J. gen. Microbiol. 30, 327–337 (1963)Google Scholar
- Suyama, Y., Munkres, K.D., Woodward, V.W.: Genetics analysis of the pyr-3 locus of Neurospora crassa: the bearing of recombination and gene conversion on inter-allelic linearity. Genetica 30, 293–311 (1959)Google Scholar
- Trotta, P.P., Burt, M.E., Haschemeyer, R.H., Meister, A.: Reversible dissociation of carbamyl phosphate synthetase into a regulated synthesis subunit and a subunit required for glutamine utilization. Proc. nat. Acad. Sci. (Wash.) 68, 2599–2603 (1971)Google Scholar
- Williams, L.G., Bernhardt, S.A., Davis, R.H.: Evidence for two discrete carbamyl phosphate pools in Neurospora. J. biol. Chem. 246, 973–978 (1971)Google Scholar
- Woodward, V.W.: Complementation and recombination among pyr-3 heteroalleles of Neurospora crassa. Proc. nat. Acad. Sci. (Wash.) 48, 348–355 (1962)Google Scholar