Abstract
Glutamine-dependent NAD(+) synthetase, Qns1, utilizes a glutamine aminotransferase domain to supply ammonia for amidation of nicotinic acid adenine dinucleotide (NaAD(+)) to NAD(+). Earlier characterization of Qns1 suggested that glutamine consumption exceeds NAD(+) production by 40%. To explore whether Qns1 is systematically wasteful or whether additional features account for this behavior, we performed a careful kinetic and molecular genetic analysis. In fact, Qns1 possesses remarkable properties to reduce waste. The glutaminase active site is stimulated by NaAD(+) more than 50-fold such that glutamine is not appreciably consumed in the absence of NaAD(+). Glutamine consumption exceeds NAD(+) production over the whole range of glutamine and NaAD(+) substrate concentrations with greatest efficiency occurring at saturation of both substrates. Kinetic data coupled with site-directed mutagenesis of amino acids in the predicted ammonia channel indicate that NaAD(+) stimulates the glutaminase active site in the k(cat) term by a synergistic mechanism that does not require ammonia utilization by the NaAD(+) substrate. Six distinct classes of Qns1 mutants that fall within the glutaminase domain and the synthetase domain selectively inhibit components of the coordinated reaction.
Highlights
Glutamine-dependent NADϩ synthetase Qns1 (1) is one of many enzymes that couples a glutamine amidotransferase (GAT)2 domain to a second active site that requires ammonia gas as a reactant (2– 4)
Experiments indicated that the synthetase and the glutaminase active sites must reside in the same polypeptide for Qns1 to function in vivo even though glutaminase mutants retained ammonia-dependent NADϩ synthetase activity, synthetase mutants retained basal glutaminase activity, and the two mutants assembled into a stable multimer in vivo (1)
Modeling and structure-based sequence analysis suggested that the ammonia channel in Qns1 is ϳ46 Å long and lined with residues conserved among glutamine-dependent NADϩ synthetases (1)
Summary
Site-directed Mutagenesis—Plasmid pB337 and derivatives were used for expression of yeast QNS1 in Escherichia coli (1). Specific activity of glutaminase without NaADϩ was determined with 20 mM glutamine and 0.9 g of Qns. The ammonia-dependent NADϩ synthetase assay was performed with 1 mM NaADϩ and 2 mM NH4Cl as an amide donor in the presence of 0.3 g of Qns. NADϩ synthewere constructed by subjecting particular single mutants to a tase activity of Qns was measured by assaying varied second round of site-directed mutagenesis. Plasmid pB177, car- concentrations of NaADϩ (0 – 400 M) in the presence of saturying QNS1 with the native promoter on a single copy HIS3 rating glutamine (20 mM). 33396 JOURNAL OF BIOLOGICAL CHEMISTRY and NADϩ synthetase activities were determined using glutamine concentrations from 0 to 10 mM in NaADϩ varying from 0 to 600 M. ATP and NaADϩ concentrations were calculated using their extinction coefficient at 259 nm (15,400 MϪ1 cmϪ1).
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