KINETIC MECHANISM OF NICOTINIC-ACID PHOSPHORIBOSYLTRANSFERASE - IMPLICATIONS FOR ENERGY COUPLING

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 nicotin ic acid (NA) and phosphoribosyl pyrophosphate (PRPP). To learn how NAP RTase uses this hydrolytic energy, we have further delineated the kine tic mechanism using steady-state and pre-steady-state kinetics, equili brium binding, and isotope trapping. NAPRTase undergoes covalent phosp horylation by bound ATP at a rate of 30 s(-1). The phosphoenzyme (E-P) binds PRPP with a K-D of 0.6 mu M, a value 2000-fold lower than that measured for the nonphosphorylated enzyme. The minimal rate constant f or PRPP binding to E-P is 0.72 x 10(5) M-1 s(-1). Isotope trapping sho ws that greater than 90% of bound PRPP partitions toward product upon addition of NA. Binding of NA to E-P.PRPP is rapid, k(on) greater than or equal to 7.0 x 10(6) M-1 s(-1), and is followed by rapid formation of NAMN and PPi, k greater than or equal to 500 s(-1). After product formation, E-P undergoes hydrolytic cleavage, k = 6.3 s(-1), and produ cts NAMN, PPi, and P-i are released. Quenching from the steady state u nder V-max conditions indicates that slightly less than half the enzym e 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 k(cat) o f 2.3 s(-1) at 22 degrees C. Energy coupling by NAPRTase involves two strategies frequently proposed for ATPases of macromolecular recogniti on and processing. First, E-P has a 10(3)-fold higher affinity for sub strates than does nonphosphorylated enzyme, allowing the E-P to bind s ubstrate from low concentration and nonphosphorylated enzyme to expel products against a high concentration. Second, the kinetic pathway fol lows ''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 s uch nonvectorial reactions an intrinsically inefficient in ATP use.