ENERGY COUPLING IN SALMONELLA-TYPHIMURIUM NICOTINIC-ACID PHOSPHORIBOSYLTRANSFERASE - IDENTIFICATION OF HIS-219 AS SITE OF PHOSPHORYLATION

Energy coupling between ATP hydrolysis and other enzyme reactions requ ires the phosphorylation of substrate-derived intermediates, or the ex istence of enzyme-derived intermediates capable of storage and transfe r of energy. Salmonella typhimurium nicotinic acid phosphoribosyltrans ferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid [Vinitsky, A., & G rubmeyer, C. (1993) J. Biol. Chem. 268, 26004-26010]. In the current w ork, we have determined that the enzyme reacts with ATP to produce a c ovalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase. We have isol ated E-P and verified its catalytic competence. E-P showed acid labili ty and base stability, diagnostic of a phosphoramidate linkage. Pyridi ne and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rat e constants consistent with published values for phosphohistidine. Two -dimensional thin-layer chromatography of alkaline-hydrolyzed E-P-32 s howed that the phosphorylated residue co-migrated with authentic 1-pho sphohistidine. Chymotrypsin and trypsin proteolysis followed by HPLC a nd peptide sequencing localized the phosphopeptide to Ala-210 to Phe-2 22 of the 399-residue protein. This peptide contains a single histidin e residue, His-219. NAPRTase phosphorylated at His-219 is an intermedi ate in the energy transduction mechanism of NAPRTase.