LIMITED PROTEOLYSIS OF SALMONELLA-TYPHIMURIUM NICOTINIC-ACID PHOSPHORIBOSYLTRANSFERASE REVEALS ATP-LINKED CONFORMATIONAL CHANGE

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) coupl es stoichiometric ATP hydrolysis with formation of nicotinate mononucl eotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrop hosphate (PRPP), Trypsin rapidly inactivated the ATPase and NAMN synth esis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protec tion against tryptic cleavage. Limited chymotryptic proteolysis of NAP RTase exhibited very similar behavior, with specific cleavage at Phe-3 82 and protection by substrates. Results suggest that a solvent-expose d 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 b y EDTA provides evidence for a distinct ATP-induced protein conformati on that acts as an intermediate in energy coupling.