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 enzyme was formed constitutively, and high activities were formed on glucose and sucrose as carbon sources. Highest activities were formed during growth on high concentrations of glucose or sucrose. The enzyme, purified about 600-fold from cell-free extracts prepared from glucose-grown mycelia, gave a double band in denaturing (SDS)-polyacrylamide gel electrophoresis. Tryptic peptide patterns suggest that the lower molecular weight band was the product of either C- or N-terminal truncation. The specific activity of the enzyme was about 40 and 35 mumol/min and mg protein with glucose and fructose as substrates, respectively. The affinity for glucose was about 10(3)-fold higher than for fructose. The subunit molecular weight was 50,000 and the molecular weight of the native protein was 100,000 by gel permeation chromatography. Of the reaction products ADP, but not glucose 6-phosphate, inhibited hexokinase activity. Citrate inhibited (K1 0.15 mM) non-competitively with respect to both glucose and ATP, which was not due to Mg(2+)-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 were comparable to the parent strain. They displayed a reduced rate of citric acid accumulation. It is concluded that the synthesis of very high hexokinase activities may counteract citrate inhibition, thereby guaranteeing a high glycolytic flux during citric acid accumulation.
Steinböck
F
F
Institut für Biochemische Technologie und Mikrobiologie, TU Wien, Vienna, Austria.
Choojun
S
S
Held
I
I
Roehr
M
M
Kubicek
C P
CP
D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D002951:Citrates / Q000378:metabolism*; D019343:Citric Acid; D006019:Glycolysis; D006593:Hexokinase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D009154:Mutation
D002951:Citrates; D019343:Citric Acid; D006593:Hexokinase