Phosphate Bound to Histidine in a Protein as an Intermediate in a Novel Phospho-Transferase System

Mammalian tissues contain a kinase involved in the intermediary metabolism of the sialic acids.1' 2 This enzyme has been extensively purified,3 studied in detail, and catalyzes the following reaction: N-Acyl-D-mannosamine + ATP Mg++ N-Acyl-D-mannosamine-6-P + ADP.To determine whether this kinase occurred in bacteria, such as Aerobacter cloacae and Escherichia coli K235,4 that metabolize N-acetyl-D-mannosamine, extracts of these organisms were examined and found to contain a novel phospho-transferase system.The system obtained from E. coli K235 consisted of two enzymes, I and II, and a histidine-containing, heat- stable protein (HPr).The sequence of reactions is: I Phosphoenolypyruvate (PEP) + HPr Mg+ Phospho-histidine- protein (P-HPr) + Pyruvate (A) P-HPr + Hexose -., -Hexose-6-P + HPr (B)PEP + Hexose M Hexose-6-P + Pyruvate (A+B)The intermediate in the system, P-HPr, is protein-bound phosphohistidine.Materials and Methods.-Unlessotherwise specified, all materials were obtained from comi- mercial sources.Previously published methods', 6 were used for the preparation, separation, and characterization of C"1-and C'4-hexosamines, N-acylhexosamines, the corresponding 6-phosphate esters, and for the periodate oxidation of the esters and the characterization of glycolaldehyde- phosphate.The following compounds were prepared as described: P-histidine,6 N-phospho- glycine,7 phosphoramidate,8 and PEP."An essential substrate for these experiments, P32-PEP was prepared enzymatically by a published procedure'0 and with the invaluable help of Dr. M. F. Utter and Mr. Douglas Kerr,-to whom we are most grateful."The P32-PEP (5-10 ,umoles per experiment) contained 200-400 c of P8a per ,umole and was purified by ion-exchange chromatog- raphy; paper chromatography and electrophoresis indicated that it was homogeneous.It was diluted with unlabeled PEP prior to use.. Purification of enzymes I, and II, and HPr: The organism, E. coli K235, was grown to the sta- cionary phase in Todd-Hewitt (Difco) broth supplemented with 1.5% glucose in a New Brunswick fermentor.Maximum yields of the phospho-transferase system were obtained when the culture was stirred during growth but without passage of air through the sparger.After washing with 1% KCl solution, the wet cell paste was stored at -18°.The cells were ruptured by sonic oscilla- tion following suspension in 0.025 M phosphate buffer, pH 7.6 (containing 0.1% 2-mercapto- ethanol and 10-3 M EDTA when enzymes I and II were desired).After centrifugation, the supernatant fluid (crude extract) was treated with charcoal to remove HPr and fractionated for I and II as outlined in Table 1.The critical step was the Qy alumina gel treatment since I was adsorbed while II was not; after washing the gel with 0.01 and 0.05 M phosphate buffers, pH 7.6, I was eluted with 0.10 M buffer.These data suggest that both enzymes were purified approximately 300-fold.Since we have not yet determined which enzyme, I or II, was present at rate-linmiting concentrations prior to their separation, the purification factor is cor- rect for only one of these enzymes, and is not known for the other.However, the availability of the purified enzymes I and II will now permit accurate analysis for each enzyme.