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A study of the oxidation of 2-Ketogluconate using cell preparations of pseudomonas aeruginosa Campbell, Lorne Arthur


Pseudomonas aeruginosa is known to dissimilate glucose by way of a pathway which does not involve phosphorylation at the hexose level. The established intermediates in this pathway are gluconate, 2-ketogIuconate, pyruvate and finally the compounds of an unconventional tricarboxylic acid cycle. The major gap in our knowledge concerns the fate of 2-ketogluconate. The enzymes responsible for the degradation of this compound have proven to be very unstable and previous attempts to obtain an active cell preparation or cell free extract have met with little success. There is some evidence that drying cells in an atmosphere of carbon monoxide preserves the enzyme in question while interfering with the complete oxidation of 2-ketogluconate. This should result in the accumulation of an intermediate product and thus would make possible the elucidation of one more step in the sequence of reactions. For this reason the work on monoxide-dried cells was continued in the hope, that they would serve as a source of the 2-ketogluconate enzyme. This technique produced a preparation with a good ability to oxidize 2-ketogluconate. However, the viscous nature of the preparation made centrifugal separation of the remaining live cells almost impossible. Glucose and gluconate grown cells when suspended in a 45% solution of sucrose and subjected to sonic vibrations produced a reproducible cell free preparation with a good ability to oxidize 2-ketogluconate. This preparation had an optimum pH of 7.4 and a respiratory quotient of 3. The mechanism of this oxidation remains unexplored. Pyruvic acid was identified as a product of this reaction. The crude sucrose sonicate was not stimulated by ATP and no phosphorylation could be detected aerobically or anaerobically by measuring acid stable (ester) phosphate. The preparation was not inhibited by 2.5 x 10⁻² sodium fluoride. Moreover, a crude sonicate of gluconate grown cells in the presence of ATP and magnesium, showed no phosphorylated compounds, by the chromatographic methods employed. The study for detection of phosphorylated compounds was carried out under strict anaerobic conditions. No new intermediates were isolated in the pathway of 2-ketogluconate breakdown and this pathway still remains unknown. However, this work has provided several methods of obtaining active cell preparations.

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