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 requires the phosphorylation of substrate-derived intermediates, or the existence of enzyme-derived intermediates capable of storage and transfer of energy. Salmonella typhimurium nicotinic acid phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid [Vinitsky, A., & Grubmeyer, C (1993) J. Biol. Chem. 268, 26004-26010]. In the current work, we have determined that the enzyme reacts with ATP to produce a covalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase. We have isolated E-P and verified its catalytic competence. E-P showed acid lability and base stability, diagnostic of a phosphoramidate linkage. Pyridine and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rate constants consistent with published values for phosphohistidine. Two-dimensional thin-layer chromatography of alkaline-hydrolyzed E-32P showed that the phosphorylated residue co-migrated with authentic 1-phosphohistidine. Chymotrypsin and trypsin proteolysis followed by HPLC and peptide sequencing localized the phosphopeptide to Ala-210 to Phe-222 of the 399-residue protein. This peptide contains a single histidine residue, His-219. NAPRTase phosphorylated at His-219 is an intermediate in the energy transduction mechanism of NAPRTase.
Gross
J
J
Department of Biochemistry and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Rajavel
M
M
Segura
E
E
Grubmeyer
C
C
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010446:Peptide Fragments; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D006639:Histidine; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; D000251:Adenosine Triphosphatases; C021864:nicotinate phosphoribosyltransferase; C005436:phosphohistidine