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J Biol Chem. 2006 Nov 3;281(44):33395-402. Epub 2006 Sep 5.

Glutamine-dependent NAD+ synthetase. How a two-domain, three-substrate enzyme avoids waste.

Author information

1
Department of Genetics and the Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.

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.

PMID:
16954203
DOI:
10.1074/jbc.M607111200
[Indexed for MEDLINE]
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