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43869-G4
Kinetic Isotope Effects on the NAD+ Synthetase-Catalyzed Reaction
The founded research program aimed to characterize kinetically and mechanistically the reaction catalyzed by NAD+ synthetase. This enzyme catalyzes the ATP-dependent transformation of nicotinic acid adenine dinucleotide (NaAD+) to NAD+, the last step in the biosynthesis of NAD+. NAD+ synthetase from M. tuberculosis and B. cereus are particularly attractive drug targets. M. tuberculosis relies on this enzyme for NAD+ biosynthesis while sporulation of B. cereus (a non-lethal strain closely related to B. anthracis) depends on NAD+ synthetase activity. These NAD+ synthetases differ in that the M. tuberculosis ortholog is a two-domain enzyme and utilizes glutamine as the nitrogen donor while the B. cereus is a simpler enzyme that uses ammonia as the nitrogen donor.
Detailed reports on the outcome of experiments carried out have been omitted in this report because they await publication. The research program proposed described kinetic isotope effects experiments to investigate the amidation reaction of the intermediate NaAD-AMP. However, almost no kinetic and mechanistic data is available on either the ammonia- and glutamine-dependent NAD+ synthetases. Expression vectors for both the B. cereus and M. tuberculosis NAD+ synthetases were constructed and conditions to obtain expressed soluble enzymes were determined. Both synthetases were purified by affinity chromatography. The fusion-tag chosen can be removed yielding the native enzyme with only one additional amino acid at the N-terminus with reasonable yields (~5mg/g wet cells). The M. tuberculosis synthetase requires an additional gel filtration step in the purification to remove aggregates. The expression and purification system chosen provides enough pure enzymes to carry out the kinetic isotope effects proposed.
We proceeded in characterizing the steady state kinetics for the reaction catalyzed by both synthetases. The glutamine-dependent enzyme displays unique characteristics. The assays used are discontinuous and extremely labor intensive. We are synthesizing a substrate analog that will allow a continuous detection of the initial rate and, therefore, ease and speed the acquisition of steady state kinetic data such as pH rate profile, characterization of metal dependence and/or substrate analogs to look for conditions likely to reveal the intrinsic isotope effect.
We have cloned in expression vector, expressed and purified all the enzymes mentioned in the research proposal necessary for the synthesis of the labeled substrates and for the measurements of the kinetic isotope effects. In addition to the enzymes mentioned, we have also cloned, expressed and purified ribokinase and PRPP synthetase. These enzymes catalyze the formation of phosphoribosyl pyrophosphate (PRPP), an expensive starting material, from ribose, a cheap precursor.
