Allosteric Properties of Bovine Liver Nicotinate Phosphoribosyltransferase

Smith, Lucile; Gholson, R.K.

doi:10.1016/s0021-9258(19)78192-7

Cited by 32 publications

(7 citation statements)

References 16 publications

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“…The existence of substrate inhibition by PP i (K I ) 320 µM) suggested that binding of PP i to E could occur and prevent ATP binding. PP i binding to E was detected (17) (1,(20)(21)(22). The mechanism of Scheme 1 suggests that stoichiometry will rise as product accumulates.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Mechanism of Nicotinic Acid Phosphoribosyltransferase: Implications for Energy Coupling

1998

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Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) is a facultative ATPase that uses the energy of ATP hydrolysis to drive the synthesis of nicotinate mononucleotide and pyrophosphate from nicotinic acid (NA) and phosphoribosyl pyrophosphate (PRPP). To learn how NAPRTase uses this hydrolytic energy, we have further delineated the kinetic mechanism using steady-state and pre-steady-state kinetics, equilibrium binding, and isotope trapping. NAPRTase undergoes covalent phosphorylation by bound ATP at a rate of 30 s-1. The phosphoenzyme (E-P) binds PRPP with a KD of 0.6 microM, a value 2000-fold lower than that measured for the nonphosphorylated enzyme. The minimal rate constant for PRPP binding to E-P is 0.72 x 10(5) M-1 s-1. Isotope trapping shows that greater than 90% of bound PRPP partitions toward product upon addition of NA. Binding of NA to E-P.PRPP is rapid, kon >/= 7.0 x 10(6) M-1 s-1, and is followed by rapid formation of NAMN and PPi, k >/= 500 s-1. After product formation, E-P undergoes hydrolytic cleavage, k = 6.3 s-1, and products NAMN, PPi, and Pi are released. Quenching from the steady state under Vmax conditions indicates that slightly less than half the enzyme is in phosphorylated forms. To account for this finding, we propose that one step in the release of products is as slow as 5.2 s-1 and, together with the E-P cleavage step, codetermines the overall kcat of 2.3 s-1 at 22 degrees C. Energy coupling by NAPRTase involves two strategies frequently proposed for ATPases of macromolecular recognition and processing. First, E-P has a 10(3)-fold higher affinity for substrates than does nonphosphorylated enzyme, allowing the E-P to bind substrate from low concentration and nonphosphorylated enzyme to expel products against a high concentration. Second, the kinetic pathway follows "rules" [Jencks, W. P. (1989) J. Biol. Chem. 264, 18855-18858] that minimize unproductive alternative reaction pathways. However, an analysis of reaction schemes based on these strategies suggests that such nonvectorial reactions are intrinsically inefficient in ATP use.

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Section: Discussionmentioning

confidence: 99%

Kinetic Mechanism of Nicotinic Acid Phosphoribosyltransferase: Implications for Energy Coupling

1998

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“…A second difference between NAPRTase and the other PRTases lies in its use of ATP. It is known that ATP stimulates NAMN formation by NAPRTase from several sources (13)(14)(15)(16)(17)(18)(19), and that stimulation is accompanied by hydrolysis of ATP stoichiometric to NAMN formed (15,(17)(18)(19). Although some of the other PRTases show allosteric stimulation by effector molecules (3), including GTP (20,21), NAPRTase is the only PRTase known to cleave the stimulator during the reaction cycle.…”

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Conversion of a Cosubstrate to an Inhibitor: Phosphorylation Mutants of Nicotinic Acid Phosphoribosyltransferase

et al. 1998

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Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.

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“…It is also not clear whether it is E-P formation, or ATP binding alone, that is required to trigger the affinity changes associated with substrate binding. The ATPase and NAMN synthesis functions of NAPRTase may not be intimately related: although in the yeast enzyme NAMN synthesis does not proceed without ATP hydrolysis (Hanna et al, 1983;Kosaka et al, 1971Kosaka et al, , 1977, in the human (Niedel & Dietrich, 1973;Smith & Gholson, 1969) and S. typhimurium (Vinitsky & Grubmeyer, 1993) enzymes uncoupled NAMN synthesis can be readily detected. In a protozoal NAPRTase, no effect of ATP is noted (Khan et al, 1967).…”

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confidence: 99%

Limited Proteolysis of Salmonella typhimurium Nicotinic Acid Phosphoribosyltransferase Reveals ATP-Linked Conformational Change

et al. 1996

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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.

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Allosteric Properties of Bovine Liver Nicotinate Phosphoribosyltransferase

Cited by 32 publications

References 16 publications

Kinetic Mechanism of Nicotinic Acid Phosphoribosyltransferase: Implications for Energy Coupling

Kinetic Mechanism of Nicotinic Acid Phosphoribosyltransferase: Implications for Energy Coupling

Conversion of a Cosubstrate to an Inhibitor: Phosphorylation Mutants of Nicotinic Acid Phosphoribosyltransferase

Limited Proteolysis of Salmonella typhimurium Nicotinic Acid Phosphoribosyltransferase Reveals ATP-Linked Conformational Change

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