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Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe

Perysinakis, A., Kinghorn, J.R., Drainas, C.
Current genetics 1994 v.26 no.4 pp. 315-320
Saccharomycetales, nitrogen metabolism, alleles, loci, glutamate dehydrogenase, nutrient availability, NADP (coenzyme), ammonium compounds, mutants, enzyme activity, amino acid metabolism, glutamic acid
The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH: EC have been determined. NADP-GDH was found to be specific for the substrates used in the reaction mixtures. NADP-GDH activity showed a sigmoidal response to changes in alpha-ketoglutarate concentrations, following Hill kinetics with a coefficient n(H) = 2. A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea. Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources. The mutants also failed to grow on allantoin alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium. Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin. In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type. Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline. The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS). Mutants were found to map in two unlinked genetic loci by recombinational analysis. One locus was designated as gdh1 and was represented by four mutant alleles (gdh1-1, gdh1-2, gdh1-3 and gdh1-4), whilst the second locus, gdh2, had one mutant allele (gdh2-1). No differences were observed in growth tests or enzymic studies between these loci.