Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate George J.G. Ruijter*, Jaap Visser Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, the Netherlands (Received 17 September 1998; accepted 19 November 1998) Abstract — Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD + , respectively. At pH 7.5 the K m for glucose 6-P was 0.48 mM, whereas the K m for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with K i values of approximately 0.2 mM and 1 μM respectively. In citric acid producing A. niger mycelium inhibition by 6-phospho- gluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production. © Société française de biochimie et biologie moléculaire / Elsevier, Paris phosphoglucose isomerase / kinetics / Aspergillus niger / erythrose 4-phosphate / 6-phosphogluconate 1. Introduction Phosphoglucose isomerase (PGI) catalyses interconver- sion of glucose 6-P (G6P) and fructose 6-P (F6P) which is the second step in glycolysis [1]. PGI has a strategic position in metabolism as several metabolic pathways branch off from the glycolytic/gluconeogenic pathway both at G6P (biosynthesis of cell wall components, oxi- dative part of pentose phosphate pathway) and F6P (mannitol biosynthesis, non-oxidative part of pentose phosphate pathway). PGI has been purified from a number of sources. The best characterized enzymes include PGI from rabbit skeletal muscle [2] and yeast [3]. Some as- pects of A. niger PGI have been studied by Singh [4], but we are not aware of any reports on PGI from other fungi. Yeast PGI is a dimeric enzyme with a subunit molecular mass of about 60 kDa [5]. PGI from most sources is inhibited by several compounds which are intermediates of central metabolism (e.g., 6-phosphogluconate, eryth- rose 4-P, ATP)[1]. In particular inhibition by erythrose 4-P (E4P) is strong, e.g., the K i value for yeast PGI is 2 μM [6]. The physiological significance of inhibition by E4P is, however, questionable, since E4P has never been detected unequivocally in metabolite extracts [7, 8]. Paoletti et al. [7] developed a sensitive assay for determi- nation of E4P, but failed to detect it in tissue extracts. One explanation could be that the level of E4P in cells is too low to be detected by this method [7, 8]. A. niger is able to accumulate a high level of citric acid from hexoses under particular conditions. The sugar substrate is metabolized mainly via glycolysis resulting in formation of pyruvate, which is then converted in a few steps to citric acid [9]. Recently, several attempts have been made to construct a model of A. niger carbohydrate metabolism in relation to citric acid production by this organism [10–12]. With such models in hand flux control analysis can be performed enabling design of a strategy to improve citric acid biosynthesis by genetic engineer- ing [11]. To support these models with reliable data we purified and characterised PGI from A. niger. Like PGI from other sources A. niger PGI is strongly inhibited by E4P. To investigate the relevance of E4P inhibition in vivo we attempted to determine E4P levels in metabolite extracts. However, since E4P could not be detected in A. niger metabolite extracts by the method of Paoletti et al. [7], we developed a novel assay for quantitative determination of E4P, based on the strong inhibition of PGI by E4P. * Correspondence and reprints Biochimie 81 (1999), 267-272 © Société française de biochimie et biologie moléculaire / Elsevier, Paris