Purification and mechanism studies on the phosphoroclastic reaction of escherichia coli
Purification of the phosphoroclastic system of Escherichia coli was undertaken. A 4-fold purification of the crude extract was obtained using heat precipitation and protamine sulfate. Some purification of Knappe's A fraction was obtained with ammonium sulfate and acetone. Evidence was obtained for the existence of two factors in the A fraction. Other purification techniques gave little success. Good evidence was obtained for the involvement of phosphotrans-acetylase in the reaction sequence. Phosphotransacetylase was purified 750-fold and characterized. The reversibility of the reaction was studied with carbon 14. Formate fixed readily into pyruvate only when pyruvate was present. Acetyl coenzyme A fixed into pyruvate also but to a much smaller degree. Better fixation without pyruvate was obtained after prior incubation and consumption of pyruvate. The dilution effect, inhibitors, effect of light, and a possible role of coenzyme B_12 were investigated. By using a deuterium label, it was shown that a hydrogen does not shift directly from the methyl group of pyruvate to formate in the reaction mechanism.
College and Department
Chemistry and Biochemistry
BYU ScholarsArchive Citation
Winkel, Cleve R., "Purification and mechanism studies on the phosphoroclastic reaction of escherichia coli" (1970). Theses and Dissertations. 8386.
Enzymes, Escherichia coli.