Enzymes of de novo purine and pyrimidine nucleotide biosynthesis are rapidly and selectively inactivated in Bacillus subtilis cells when they are starved for a nutrient and begin to sporulate. This project is focussed on learning the biochemical mechansim of the inactivation of two of these enzymes: aspartate transcarbamylase and flutamine phosphoribosylpyro-phosphate amidotransferase, both of which catalyze initial reactions of their respective pathways. A second objective is to learn the physiological and biochemical processes which regulate these inactivation processes. Immunochemical experiments have shown that both enzymes are probably rapidly degraded during or after inactivation. Thus, the research aims at a clarification of the mechanism and regulation of selective intracellular proteolysis in a bacterium which lacks a lysosomal compartment. The degradation of aspartate transcarbamylase has been shown to require metabolic energy, while inactivation of amidotransferase specifically requires O2. In the latter case the role of O2 appears to involve oxidation of a Fe4S4 center in the enzyme. The discovery of such a center in a non-redox enzyme opens a new area in the biochemistry of iron-sulfur proteins. Proposed research involves further study of the inactivation processes by vitro reconstruction, immunochemical, and physiological studies. Synthesis of both enzymes is abruptly shut off prior to the beginning of their inactivation. The mechanism of this selective change in gene expression is of considerable interest to the study of cellular differentiation. Experiments are in progress to isolate and clone the DNA coding for aspartate transcarbamylase, so that its transcription in vivo and in vitro can be examined.