Abstract
Abstract An enzyme, phosphoramidate-ADP phosphotransferase, has been isolated from dried bakers' yeast. We have previously shown that phosphoramide, N-phosphorylglycine, and N-phosphohistidine can act as phosphate donors. Phosphoiodohistidine was shown also to function as a phosphate donor and the enzyme was stereospecific for the l form of this compound. The following compounds acted as phosphate acceptors in this system in decreasing order of activity: ADP, dADP, GDP, CDP, TDP, dCDP, TDP, UDP. When phosphoramide was used as a donor, the pH maximum was 7.0 to 7.2. When phosphoiodohistidine was used as the donor, a double pH maximum with peaks at 6.9 and 7.8 was noted. The Arrhenius energy of activation was determined to be 12.2 kcal per mole. Apparent Km values were 1.15 x 10-4 m for ADP and 1.39 x 10-4 m for phosphoramide. Mg++ ions were not required and were found to be inhibitory with either phosphoiodohistidine or phosphoramide as the donor. A wide variety of other possible inhibitors was checked. Compounds which affect sulfhydryl groups on enzymes were shown to inhibit this enzyme. Known inhibitors of oxidative phosphorylation, oligomycin and sodium azide, were also shown to inhibit enzymatic activity. However 2,4-dinitrophenol had no effect. With the use of 32P-labeled phosphoramide, an enzyme-32P-protein was isolated by diethylaminoethyl cellulose chromatography. This intermediate was capable of transfering labeled phosphate to ADP to form AT32P.
Highlights
We have previously shown that phosphoramide, N-phosphorylglycine, and
Phosphoiodohistidine was shown to function as a phosphate donor and the enzyme was stereospecific for the L form of this compound
The following compounds acted as phosphate acceptors in this system in decreasing order of activity: ADP, dADP, GDP, CDP, TDP, dCDP, TDP, UDP
Summary
Phosphoramidate-ADP phosphotransferase, has been isolated from dried bakers’ yeast. The supernatant fluid was treated with ammonium sulfate to 407; saturation and the precipitate was removed by ccntrifugation Blthough this protein pellet contained some activity it was usually discarded. The result,s of this partial purification appear in Table Il. The apparent recovery of over lOO?i of the enzyme activity can be explained by the removal of some of the contaminating phosphatases and adenylate kinase and because ammonium sulfate was shown to activate phosphoramidate-ADP-phosphotransferase and to inhibit adenylate kinase (31). (Th is solution has the following composition: 120 g of naphthalene, 70 ml of liquifluor (Nuclear Chicago), and dioxane to 1000 ml.) The reaction mixture for determining enzyme activity n-as as follows: 0.25 ml of 0.03 M ADP, 0.25 ml of 0.03 M WP, 0.25 ml of enzyme solution, and 0.75 ml of 0.05 M Tris maleate buffer, pH 7.2 This mixture was allowed to react at room temperature for 30 min unless otherwise specified. Of the ?Vl solution n’as count,ed and the counts per min per pmole lverc determined
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