Properties of a particulate chitin synthetase from Mucor rouxii.

The properties and behavior of a “microsomal” (100,000 X g particles) chitin synthetase of Mucor rouxii were investigated. The enzyme utilizes uridine diphosphate N-acetylD-glucosamine (UDP-GlcNAc) as glycosyl donor and is strongly and specifically activated by free N-acetyl-o-glucosamine (GlcNAc). A variety of GlcNAc analogues were tested as activators but were found ineffective. A small proportion of free GlcNAc was incorporated into chitin; however, the activation of chitin synthetase by GlcNAc is probably an allosteric effect. Seemingly, UDP-GlcNAc is also a positive allosteric effector of the enzyme. Diacetylchitobiose was also synthesized in the reaction mixtures. However, the kinetics of its formation do not support the proposal that this dimer is an intermediate in chitin synthesis. Added primer was not essential for the operation of the microsomal chitin synthetase. Nevertheless, addition of higher Nacetylchitodextrins produced large stimulations indicative of primer shortage in the particles. Certain lipid extracts of IM. rouxii proved to be highly stimulatory for chitin synthesis. However, efforts to obtain evidence for a lipid intermediate proved negative. Chitin synthetase was also stimulated by ATP, CTP, and GTP but UDP was inhibitory. Optimum pH was about 6.5. The enzyme was exceedingly susceptible to Mg++ concentration. Microsomal chitin synthetase was relatively stable in the frozen state if suspended in phosphate buffer but not in Tris buffer. Mitochondrial (10,000 x g) enzyme, by contrast, rapidly decayed under similar conditions.