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Biochemistry

Kinetic mechanism of nicotinic acid phosphoribosyltransferase: implications for energy coupling.


PMID 9521741

Abstract

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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N7764
Nicotinic acid mononucleotide
C11H14NO9P