The significance of amino acid inhibition of NADP-linked glutamate dehydrogenase in the physiological control of glutamate synthesis in Candida utilis.

Abstract

Summary: Isotopic nitrogen (15N) was used to measure the rate of synthesis of glutamic acid and of glutamine in turbidostat cultures of Candida utilis grown on ammonium phosphate as N source. Steady-state experiments revealed that glutamic acid exists in more than one metabolic pool; approximately 5 % of the free amino acid is in a separate ‘storage’ pool while a small but measurable component is apparently in a separate pool used specifically for the synthesis of glutamine. Over the temperature range 15 to 25 °C the proportion of total nitrogen assimilated through glutamate was constant at 75 %; 12 to 15 % entered cellular metabolism through glutamine amide. Decrease of ammonium levels in the culture medium resulted in slightly lower growth rates and a decreased size of glutamate pool, but the proportion of nitrogen assimilated through glutamate remained constant. Steady-state conditions could be disturbed by withholding nitrogen for a short period (10 to 20 min) or by rapid shift of temperature. In both cases transient changes in growth rate, amino acid pools and rate of synthesis of glutamate and glutamine occurred. The rate of glutamate synthesis was very closely correlated with the size of the total pool of amino acids, and in vitro experiments confirmed that amino compounds were allosteric inhibitors of NADP-glutamate dehydrogenase. The in vivo data yielded a Hill number of 8, indicative of the very sharp inhibition occurring over the range 0·15 to 0·25 m-amino acids. For extracted enzyme preparations the Hill number was smaller (2 to 6); this may imply partial dissociation at the lower protein concentrations used for in vitro experiments. Analyses of organisms grown under steady-state conditions at various temperatures revealed that the amino acid feedback control on glutamate dehydrogenase is probably the chief mechanism involved in the regulation of glutamate synthesis to match growth requirements at different temperatures. Other physiological conditions in which glutamate formation is controlled are briefly discussed.