The Stereochemical Course of Nucleotidyl Transfer Catalysed by NAD Pyrophosphorylase

Abstract

NAD pyrophosphorylase catalyses nucleotidyl transfer from adenosine (R)-~'- [E-' 70]triphosphate to nicotinamide mononucleotide with inversion of configuration at the a-P giving (S)- ['70]NAD'. The simplest interpretation of this observation is that the adenylyl group is transferred directly from ATP to the co-substrate by an 'in line' mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD ' regio-specifically at the adenylyl terminus of the pyrophosphate bond.NAD pyrophosphorylase catalyses nucleotidyl transfer from MgATP to nicotinamide mononucleotide (NMN) to form NAD' [I].This reaction is the final step in the main biosynthetic pathway to NAD' in eukaryotic cells [2] and it is particularly noteworthy that NAD pyrophosphorylase is the only enzyme in the pathway that is located in the cell nucleus [3 -51. It has been suggested that the nuclear location of NAD pyrophosphorylase is related to the involvement of NAD+ in the regulation of cellular activity [6] and a correlation between NAD pyrophosphorylase activity and mitosis has been observed [7, 81; there are also numerous examples of a correlation between NAD pyrophosphorylase activity and DNA synthesis [4,. This suggestion was supported by the discovery of a chromosomal protein, poly(ADP-ribose) synthetase [13 -151, which uses NAD' as a substrate for the post-translational modification of nuclear proteins [16,17], including histones [I 8, 191. Although the precise physiological role of this enzyme remains unclear, there is evidence that poly(ADP-ribosy1)ation is involved in DNA replication, mitosis, transcription and the repair of damaged DNA [17, 20-231.NAD pyrophosphorylase activity is very low in tumour cells when compared with normal adult cells and is probably close to the critical level for cell survival [4, 81. Consequently NAD pyrophosphorylase has been regarded as a potential target site for cancer chemotherapy [S], since the minimum inhibitory concentration lethal to tumour cells is not likely to cause serious damage to normal cells. Since a knowledge of the mechanism of action of this enzyme and the juxtaposition of the substrates when bound to the enzyme would undoubtedly assist in the design of a specific inhibitor, we have investigated the stereochemical course of nucleotidyl transfer catalysed by .NAD pyrophosphorylase.N AD pyrophosphorylase catalyses exchange between ATP and [32P]PPi in the presence of NMN; in the absence of NMN Enzyme. NAD pyrophosphorylase or NMN adenylyltransferase (EC 2.7.7.1).only a small amount of exchange occurs and the reasonable conclusion was made that there is no support for an adenylylenzyme intermediate [24]. However the possibility of an induced conformational change of the enzyme on binding NMN leading to a more facile adenylyl transfer to the putative adenylyl-enzyme intermediate is difficult to rule out [25]. We have undertaken therefore to determine the stereochemical course of the nucleotidyl transfer catalysed by NAD pyrophosphorylase using adenosine (R)-5'-[a-170]triphosphate [26,...

Enzymology of Nad + Synthesis

“…NMN adenylyltransferase activity, which is considered critical for cell survival (Hughes et al, 1983), is very low in tumor cells, representing a potential target for cancer chemotherapy (Morton, 1958;Emanuelli et al, 1995). The kinetic study results obtained for the eukaryotic enzyme are consistent with an ordered substrate addition and product release , in agreement with the stereochemical analysis conducted by Lowe and Tansley (1983). However, improved understanding of the mechanism of action of this enzyme could undoubtedly be helpful in the design of a specific inhibitor as a candidate drug for anticancer chemotherapy.…”

Enzymology of NAD+ homeostasis in man

“…Experimental data seem to indicate a similarity with respect to the cations requirement between human NMNAT-1 and its thermophilic counterpart, whereas yeast isoenzymes exhibit maximal activity with Ni 2+ and Co 2+ [102 -104]. Initial-rate kinetic studies and product inhibition patterns, obtained analyzing the reaction on both directions [94], showed that the NMNAT-1 catalytic mechanism is ordered Bi-Bi in agreement with the stereochemical data [105]. Compared with the eubacterial enzyme, which appears to prefer NaMN over NMN, human NMNAT-1 seems to use both mononucleotides with the same efficiency, thus being capable of participating in both de novo and salvage pathways of NAD + synthesis [95].…”

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

“…Because there are few interactions with the enzyme that would stabilize the NMN phosphate in its bent conformation, we assume that this conformation is less favorable or less stable than the "extend-ed" conformation. The alternative bent and extended NMN conformations in the PNAT active site provided critical insight into the catalytic mechanism of PNAT, which has been proposed to use an in-line nucleophilic attack of the NMN phosphate on the ATP ␣-phosphate (47). The propensity of NMN to adopt an extended conformation would bring the NMN phosphate close to the ATP ␣-phosphate for such an attack.…”