UBC Theses and Dissertations
A study of the pathways of glucose oxidation of Pseudomonas aeruginosa Reid, K. Garth
An effort has been made to demonstrate that the major pathway for glucose oxidation in Pseudomonas aeruginosa (ATCC 9027) involves the sequence of reactions: glucose →gluconate → 2-ketogluconate → 2-keto-6- phosphogluconate → 6-phosphogluconate. It appears however, that extracts of this organism are capable of phosphorylating glucose directly, that is, to yield glucose-6-phosphate and subsequently 6-phosphogluconate. A study of this latter pathway was felt to be necessary in order to evaluate the likelihood of it being a major alternative to the established non-phosphorylated pathway. Since it is known that glucose-6-phosphate dehydrogenase from P. aeruginosa and other microorganisms as well as from certain animal tissues exhibits a marked sensitivity to various nucleotides particularly to adenosine triphosphate. A study of this inhibition was made in order to assess the possible role that this sensitivity may play in determining the importance of this pathway as the major route of glucose oxidation. Enzyme fractionation studies revealed that hexokinase and glucose-6-phosphate dehydrogenase could be separated either by an ethanol fractionation or by an alkaline ammonium sulfate fractionation. The best separation of dehydrogenase was obtained using ethanol although hexokinase could only be isolated using the alkaline ammonium sulfate method. Cell free extracts of P. aeruginosa oxidize glucose to 2-keto-gluconate but carry the reaction no further. This represents a consumption of l µM of oxygen per µM glucose. In the presence of ATP the amount of oxygen consumed was reduced to a maximum of 0.5 µM per µM glucose, indicating the accumulation of a compound less oxidized than 2-ketogluconic acid. 6-phosphogluconate appeared to conform to the requirements of such a compound. Chromatographic analysis of reaction mixtures containing ATP revealed the accumulation of a phosphorylated compound which could not be identified. Under in vitro conditions both pathways appear to be operable but the non-phosphorylated pathway accounts for most of the glucose in the metabalizing organism.
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