PHOSPHOHISTIDYL ACTIVE-SITES IN POLYPHOSPHATE KINASE OF ESCHERICHIA-COLI

In the synthesis of inorganic polyphosphate (polyP) from ATP by polyph osphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked ph osphoenzyme was previously identified as the intermediate. The phospha te is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N-3-phosphohistidine. Tryptic digests of [P-32]PPK contain a predominant P-32-labeled peptide that includes His-441. Of the 16 hist idine residues in PPK of E. coli, 4 are conserved among several bacter ial species, Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, wh ereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes.