Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.
Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.
Sorci
Leonardo
L
Istituto di Biotecnologie Biochimiche, Università Politecnica delle Marche, Via Ranieri 67, 60131 Ancona, Italy.
Cimadamore
Flavio
F
Scotti
Stefania
S
Petrelli
Riccardo
R
Cappellacci
Loredana
L
Franchetti
Palmarisa
P
Orsomando
Giuseppe
G
Magni
Giulio
G
D002460:Cell Line; D045744:Cell Line, Tumor; D002712:Chlorides / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D009243:NAD / Q000096:biosynthesis*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000737:chemistry; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011726:Pyridinium Compounds; D012254:Ribavirin / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D007527:Isoenzymes; D011726:Pyridinium Compounds; D017967:Zinc Compounds; C520355:tiazofurin monophosphate; D015636:Magnesium Chloride; C018613:nicotinamide-beta-riboside; D009243:NAD; D009536:Niacinamide; D012254:Ribavirin; C016837:zinc chloride; C484007:NMNAT1 protein, human; C484008:NMNAT2 protein, human; C484009:NMNAT3 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase