| Nicotinamide adenine dinucleotide (NAD+) is a crucial metabolic cofactor that along with its phosphorylated counterpart, nicotinamide adenine dinucleotide phosphate (NADP+), participates in over 300 known enzyme-mediated reduction/oxidation reactions. NAD+ synthetase (E. C. 6.3.5.1) from Bacillus subtilis catalyzes the final step in the biosynthesis of NAD+. In this reaction, the nicotinic acid adenine dinucleotide (NaAD) carboxylate carbon is activated toward nucleophilic attack through the formation of an NAD+ adenylate. NAD+ synthetase from B. subtilis was shown to be an NH3-dependent form of this enzyme. To explore the putative NH4+-binding site, a liquid chromatography-mass spectrometry (LC-MS) based end-point assay was developed. We demonstrated the existence of an NH4+-specific site in this enzyme that is capable of binding other small amine cations and using the cations as alternative nucleophiles. With molecular modeling studies, we proposed that both cation-binding sites reported by Rizzi et al. (Rizzi M., Bolognesi, M., and Coda, A. (1998), Structure, 6,1129–1140.] could stabilize amine cations and that both could reproduce the observed specificity trends. To further understand the kinetic mechanism of this enzyme, steady-state kinetic studies were performed. These studies indicated that this enzyme follows a Bi Uni Uni Bi Ping Pong kinetic mechanism.{09}In the first step of the reaction a MgATP (adenosine triphosphate) and NaAD bind and react to form the adenylate, with the release of MgPPi (pyrophosphate). In the second step, NH4 + binds, deprotonates, and attacks the reactive NAD+ adenylate, releasing adenosine monophosphate (AMP) and NAD+ in order. We demonstrated that Mg2+ activates the enzyme-mediated reaction through the formation of a MgATP2− complex, which acts as the true substrate, and that a second Mg2+ further enhances enzyme activity through binding to a modifier site. These ions were implicated in both weakening the α-β phosphate bond and in ordering the major loop 204–225, which was shown to order with the binding of MgATP. We postulate that the observed mechanism is a direct result of these structural changes. We conducted a series of inhibitor and alternative substrate studies to investigate the specificity of the mononucleotide site. From these data, we propose that specificity arises from a mixture of interactions with the triphosphate tail, the ribose diol groups, and the adenine ring. |
