Deacetylation of N-acetyl-L-glutamic acid by Neurospora crassa.

Photosynthetic bacteria and blue-green algae synthesize ornithine from glutamic acid by a cyclic route in which the intermediate N-acetylglutamic acid is regenerated by a transacetylation reaction; furthermore, in these microorganisms, arginine, which is synthesized from ornithine, is a specific inhibitor of N-acetyl-,y-glutamokinase which phosphorylates N-acetylglutamic acid on its y-carboxyl group (D. S. Hoare and S. L. Hoare, J. Bacteriol. 92:375, 1966). Enteric bacteria, however, lack the transacetylase and contain an N-acetyl--y-glutamokinase which is insensitive to arginine. In the course of a preliminary survey of the distribution of the transacetylase and the "arginine-sensitive" N-acetyl-y-glutamokinase among microorganisms, extracts of the fungus Neurospora crassa were examined and were found to contain an enzyme which catalyzed the deacetylation of N-acetylglutamic acid. The presence of the deacetylase was first indicated in qualitative experiments by use of dialyzed cell-free extracts of N. crassa to demonstrate the transacetylation reaction between Nacetylglutamate and ornithine. Paper electrophoresis was used to resolve the reaction products. The formation of Na-acetylornithine and glutamate was taken to indicate the presence of the transacetylase. However, controls in which Nacetylglutamate was incubated with extracts in the absence of ornithine also showed the formation of free glutamate. The rapid deacetylation of N-acetylglutamate also frustrated efforts to demonstrate the presence of an arginine-sensitive N-acetyl-y-glutamokinase in N. crassa. Thus, although an arginine sensitive N-acetyl--y-glutamokinase and transacetylase were readily demonstrated in extracts of the eucaryotic alga Chiamydomonas reinhardii, it was not possible to determine whether an arginine-sensitive Nacetyl-'y-glutamokinase was present in N. crassa. (Table 1).The assay, depending on the formation of a hydroxamate from N-acetylglutamyl--yphosphate resulted in a slow arginine-insensitive reaction. Slow formation of a hydroxamate derivative under these conditions might be due to the action of -y-glutamokinase (involved in proline biosynthesis) on glutamate produced by the deacetylation of N-acetylglutamate. Evidence in support of the deacetylation of N-acetyl-L-glutamate is given in Table 2: tDialyzed extracts of N. crassa were incubated with N-acetyl-L-glutamate or N-acetyl-DL-glutamate at pH 7.0 and 35 C, and the reaction was stopped by