Abstract
The regulation of the enzyme cytosine deaminase was investigated at the level of enzyme synthesis and at the level of enzyme activity in Salmonella typhimurium. Regulation of cytosine deaminase synthesis was examined to determine whether control of gene expression was present in S. typhimurium. From the experiments performed, regulation of enzyme synthesis appeared to exist. By utilizing selected strains of S. typhimurium, it is possible to exclude induction of cytosine deaminase synthesis as the mode of regulation. Rather, cytosine deaminase synthesis appeared to be repressed by pyrimidine compounds. More specifically, uracil-related compounds appeared to be the primary repressing metabolites while cytosine-related compounds appeared to occupy a secondary role in the regulation. In summary, repression of cytosine deaminase synthesis by pyrimidines was evident and may indicate an anabolic role for the enzyme. In order to determine the regulation at the level of enzyme activity, cytosine deaminase was partially purified from a S. typhimurium strain. Cytosine deaminase was partially purified 419-fold from crude extract utilizing streptomycin-heat treatment, ammonium sulfate fractionation, and hydrophobic interaction chromatography. Disc gel electrophoresis indicated that the final enzyme preparation was impure. The pH optimum of the partially purified enzyme was determined to be 7.5. Its temperature optimum of 45-50°C indicated that cytosine deaminase was rather thermostable. With regard to substrate specificity, the enzyme was capable to rapidly deaminating cytosine. 5-Fluorocytosine was deaminated quite slowly while the enzyme was incapable of deaminating 5-methylcytosine. Classical Michaelis-Menten kinetics were exhibited by the cytosine deaminase reaction and an apparent Km of 0.77 mM for cytosine was determined from a Lineweaver-Burk plot. A variety of compounds were tested as possible effectors of cytosine deaminase in S. typhimurium. The most effective inhibitor of the enzyme was orotidine monophosphate (OMP). Pure non-competitive inhibition of the reaction was observed using a Lineweaver-Burk plot. Only the maximal velocity of the reaction was affected by the addition of OMP. Pyrophosphate was determined to be the most effective activator of the cytosine deaminase reaction...
West, Thomas Patrick (1980). The regulation of cytosine deaminase in Salmonella typhimurium. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -676388.