Glutamine-dependent NAD+ synthetase. How a two-domain, three-substrate enzyme avoids waste.
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.
Wojcik
Marzena
M
Department of Genetics and the Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Seidle
Heather F
HF
Bieganowski
Pawel
P
Brenner
Charles
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019732:Amide Synthases / Q000145:classification / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism; D001665:Binding Sites; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity
D005973:Glutamine; D009243:NAD; C018348:nicotinic acid adenine dinucleotide; D000641:Ammonia; D000255:Adenosine Triphosphate; D019732:Amide Synthases; C038479:NAD+ synthase