UBC Theses and Dissertations
A study of the aconitase enzyme system of Psuedomona aeruginosa Duerksen, Jacob D.
Earlier work on aconitase has resulted in the development of the concept that a single enzyme catalyzed the reactions: citrate ⇌ cis-aconitate ⇌ isocitrate. Preliminary work with Pseudomonas aeruginosa, indicated that in this organism two or more enzymes are required for the conversion of citrate to isocitrate. The present study was undertaken in an effort to clarify the status of aconitase in this organism. Methods for the accurate determination of citrate, cis-aconitate, and isocitrate were investigated. Citrate was readily determined by a pentabroraoacetone procedure. No satisfactory method for cis-aconitate was found because of the interference of cysteine and Fe⁺⁺ present in the reaction mixture. Isocitrate was determined with pig heart isocitrate dehydrogenase. Estimation of these three tricarboxylic acids by column and paper chromatography was not achieved. Column chromatography usually resulted in separation, but not in quantitative recovery of these acids, whereas no separation was obtained with paper chromatography. Purification must be effected in order that the characteristics of aconitase may be studied more accurately. Ammonium sulphate precipitation and protamine sulphate treatment were used in attempts to purify a cell-free extract of P. aeruginosa. Little success was attained in these purifications because of the instability of the enzymes, especially that fraction catalyzing isocitrate formation from cis-aconitate. No activity of this reaction was maintained beyond the preliminary purification, whereas activity of citrate formation from cis-aconitate was maintained through several steps. Studies on the rate of formation of citrate and isocitrate from cis-aeonitate by a partially purified system were not conclusive .because of the presence of other enzymes causing the anaerobic disappearance of the three tricarboxylic acids. However, these rate studies indicated that cysteine and Fe⁺⁺ are necessary for optimum formation of citrate from cis-aconitate, but not for isocitrate formation. It appears that a second factor is necessary for optimum isocitrate formation. A more complete purification is necessary to verify these preliminary conclusions and to ascertain the status of the number of enzymes present in the aconitase enzyme system.
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