Synthesis of chitin by particulate preparations from Aspergillus flavus

Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6-7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg++. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20 mM. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 muM) and (Ki 1.35 mM), the latter giving complex kinetics.