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

Abstract

The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH:EC 1.4.1.4) 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 nH = 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).(ABSTRACT TRUNCATED AT 250 WORDS)