[1, 2‐ 13 C]‐Acetate metabolism in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert 2‐oxoglutarate and glutamate. In yeast, NADP‐dependent enzymes, encoded by GDH1 and GDH3 , are reported to synthesize glutamate from 2‐oxoglutarate, while an NAD‐dependent enzyme, encoded by GDH2 , catalyzes the reverse. Cells were grown in nonfermentable acetate/raffinose (Ace/Raf) to examine the role(s) of these enzymes during aerobic metabolism. The doubling time of wt, gdh2Δ , and gdh3Δ cells was comparable at ~ 4 hours. NADP‐dependent GDH activity (Gdh1p + Gdh3p) in wt, gdh2Δ , and gdh3Δ was decreased ~80% and NAD‐dependent activity (Gdh2p) in wt and gdh3Δ was increased ~20‐fold in Ace/Raf as compared to glucose. Cells carrying the gdh1Δ allele did not grow in Ace/Raf, yet both the NADP‐dependent (Gdh3p) and NAD‐dependent (Gdh2p) GDH activity was ~3‐fold higher. Metabolism of [1, 2‐ 13 C]‐acetate and analysis of carbon NMR spectra was used to examine glutamate metabolism. Analysis of 13 C‐isotopmers and enrichment of glutamate carbons indicate a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wt. Enrichment of 13 C of glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at < 15% of wt. These data suggest that Gdh1p is the primary GDH enzyme, even under nonfermentative conditions, and Gdh2p and Gdh3p play a noticeable role in glutamate metabolism. (Supported by NIH grant GM069372 [P. J. T.])