Limited proteolysis of Salmonella typhimurium nicotinic acid phosphoribosyltransferase reveals ATP-linked conformational change.
Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.
Rajavel
M
M
Department of Biochemistry and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Gross
J
J
Segura
E
E
Moore
W T
WT
Grubmeyer
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015870:Gene Expression; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012486:Salmonella typhimurium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; D014357:Trypsin; C021864:nicotinate phosphoribosyltransferase