Salvage pathway for NAD biosynthesis in Brevibacterium ammoniagenes: regulatory properties of triphosphate-dependent nicotinate phosphoribosyltransferase.
As the rate-limiting enzyme, catalyzing the first reaction in NAD salvage synthesis, nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) is of important interest for studies of intracellular pyridine nucleotide pool regulation. We have purified NAPRTase 520-fold from Brevibacterium ammoniagenes ATCC 6872 without using an over-expression system by applying acid treatment, salt fractionation, Ca-phosphate gel treatment, anion exchange column chromatography and size-exclusion gel filtration. Unlike this enzyme from other sources, B. ammoniagenes NAPRTase was found to be controlled by the feedback inhibition by the end product NAD with K(i)=0.7+/-0.1 mM. The reaction products, pyrophosphate and nicotinate mononucleotide, also decreased the enzyme activity, as did other intermediates of NAD synthesis, such as AMP, ADP and a NAD direct precursor, nicotinate adenine dinucleotide or deamido NAD. The enzyme was observed to require a nucleoside triphosphate for its activity and showed the maximum affinity for ATP. The specificity, however, turned out to be poor, and ATP could be substituted by other nucleoside triphosphates as well as by sodium triphosphate. The kinetic characteristics of the enzyme are reported. For the first time, our data have experimentally revealed such complicated stimulatory and inhibitory effects by the intermediates of NAD biosynthesis on one of its salvage enzymes, NAPRTase. On the basis of these data, the key role of NAPRTase is discussed in light of the regulation of NAD metabolism in B. ammoniagenes.
Dulyaninova
N G
NG
A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, 33 Leninsky Prospect, 117071, Moscow, Russia. inbio@glas.apc.org
Podlepa
E M
EM
Toulokhonova1
L V
LV
Bykhovsky
V Y
VY
D000225:Adenine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D001951:Brevibacterium / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D009243:NAD / Q000096:biosynthesis*; D009525:Niacin / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011122:Polyphosphates / Q000494:pharmacology; D011726:Pyridinium Compounds / Q000494:pharmacology
D011122:Polyphosphates; D011726:Pyridinium Compounds; D009243:NAD; D009525:Niacin; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; C021864:nicotinate phosphoribosyltransferase; D000225:Adenine; C005692:triphosphoric acid