DEAMIDATION OF HPR, A PHOSPHOCARRIER PROTEIN OF THE PHOSPHOENOLPYRUVATE - SUGAR PHOSPHOTRANSFERASE SYSTEM, INVOLVES ASPARAGINE 38 (HPR-1) AND ASPARAGINE 12 (HPR-2) IN ISOASPARTYL ACID FORMATION

Histidine-containing protein, HPr, of the phosphoenolpyruvate: sugar p hosphotransferase system in Escherichia coli, when incubated at elevat ed temperatures forms many species of protein. The two major species a re HPr-1 and HPr-2, which have been shown to lack one or two amides, r espectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (19 71) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 i s shown to be pH-dependent and does not occur readily below pH 6. Inve stigation of the identities and properties of the two residues that de amidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation spe cies by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor a ctivities; characterization of tryptic and VS-protease peptides; obtai ning two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidatio n of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. H Pr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamida tion. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, resi dues that may be involved in the active site, had impaired phosphohydr olysis properties and decreased phosphoenolpyruvate:sugar phosphotrans ferase system activity.