CHARACTERIZATION AND REGULATORY PROPERTIES OF A SINGLE HEXOKINASE FROM THE CITRIC-ACID ACCUMULATING FUNGUS ASPERGILLUS-NIGER

A single glucose-phosphorylating enzyme has been detected and purified from the citric acid accumulating fungus Aspergillus niger. The enzym e was formed constitutively, and high activities were formed on glucos e and sucrose as carbon sources. Highest activities were formed during growth on high concentrations of glucose or sucrose. The enzyme, puri fied about 600-fold from cell-free extracts prepared from glucose-grow n mycelia, gave a double band in denaturing (SDS)-polyacrylamide gel e lectrophoresis. Tryptic peptide patterns suggest that the lower molecu lar weight band was the product of either C- or N-terminal truncation. The specific activity of the enzyme was about 40 and 35 mu mol/min an d mg protein with glucose and fructose as substrates, respectively. Th e affinity for glucose was about 10(3)-fold higher than for fructose. The subunit molecular weight was 50 000 and the molecular weight of th e native protein was 100 000 by gel permeation chromatography. Of the reaction products ADP, but not glucose 6-phosphate, inhibited hexokina se activity. Citrate inhibited (K-I 0.15 mM) non-competitively with re spect to both glucose and ATP, which was not due to Mg2+-chelation. 2- Deoxyglucose resistant mutant strains of A. niger were isolated which showed decreased growth rate and activity of hexokinase during growth on glucose, while their growth on fructose and hexokinase activities w ere comparable to the parent strain. They displayed a reduced rate of citric acid accumulation. It is concluded that the synthesis of very h igh hexokinase activities may counteract citrate inhibition, thereby g uaranteeing a high glycolytic flux during citric acid accumulation.