Glutamine-dependent NAD+ Synthetase HOW A TWO-DOMAIN, THREE-SUBSTRATE ENZYME AVOIDS WASTE

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 kcat 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.