Enzymatic alcoholyses of NAD: A New type of ADP‐ribosylation reaction catalysed by NAD glycohydrolase

Abstract

As part of studies concerning the transglycosidation action substrates in aqueous media and upon their inhibitory effect of NAD glycohydrolase, the hydrolase-catalysed reaction of on the enzyme activity. 'H NMR analyses demonstrated the normal alkyl alcohols (six alcohols from methanol to 1-hexa-alkyl P-riboside structure of compounds 1-6 and the nonnol) has been investigated. All the alcoholic substrates were equivalence of the 0-methylene protons of their alkoxy found to undergo ADP-ribosylation with varying efficiences, groups. This is the first report of characterization of O-ribosyto give the corresponding 0-ribosylated products (1 -6). lated products formed by the transglycosidation action of Yields (1 1-67%) were dependent upon the solubility of the NAD glycohydrolase.In recent years, NAD glycohydrolase (NADase) [EC 3.2.2.51 has been the subject of several studies aimed at elucidating its functional It has been reported that various mammalian NADases show bifunctional activities (P-NAD hydrolysis and ADP-ribose tran~fer) [~-~]. Meanwhile, CD38, a blood cell surface antigen has been found to exhibit not only the glycohydrolase activity of NAD, but also ADP-ribosyl cyclase activity whereby it reversibly converts NAD into cyclic ADP-ribose, a new second messenger involved in the regulation of Ca2+ channels in mammalian ceII~[~~*l.We have previously studied the transglycosidation action of pig brain NADase and reported nitrogen-containing heterocyclic molecules as novel substrates there~fI~-'~]. In such cases, when the substrates were suitably modified, the transglycosylated substrates obtained could be utilized as key intermediates for the stereospecific synthesis of naturally occurring nucleotides or nu~leosides [~~'~]. On the other hand, Yost and Anderson[131 have examined the activity of NADase derived from Bungarus jacatus (poisonous snake) venom and showed indirect biochemical evidence for its ADP-ribosyltransferase-like action on some straight-chain alkyl alcohols. However, they neither isolated nor characterized the alcoholysis products of NAD. Thus, it is of considerable interest to ascertain whether a mammalian NADase similarly catalyses an ADP-ribose transfer from NAD to the alcohols, from the viewpoint of understanding the functional roles of NADase.In this paper, we describe a new type of pig brain NADase-catalysed ADP-ribosylation of some primary alkyl alcohols and the 'H NMR spectroscopic characterization of the ADP-ribosylated alcohols thus obtained.For this study, six primary alcohols (methanol, ethanol, 1-propanol, 1 -butanol, 1-pentanol and 1 -hexanol) were selected as substrates of NADase: Secondary and tertiary alcohols were found to be unsuitable as substrates, probably as a result of steric factors. In preliminary pilot tests, methanol and ethanol showed no detectable inhibitory effect on NADase activity at concentrations up to 2 M. I-Propanol, however, appreciably inhibited the enzymatic activity under the same conditions. As for other three alcohols, solubility in water decreases rapid...

Unusual Enzymatic Hydrolysis of NAD by Solubilized Form of NAD+ Glycohydrolase.

“…Even in the presence of ethanol, one of the most efficient substrates for the transglycosylational action of NADase, 5) no substantial transglycosylated ethanol which was available in good yield in the parent NAD/pNADase system, was produced. This unexpected catalytic alteration of sNADase could be caused by its possible stereoconformational change which was attributed to dissociation from a membrane anchor.…”

“…There have been many reports [1][2][3][4][5][6][7][8][9] concerning substrates for the transferaselike activity of NADase, including synthetic low molecular compounds [2][3][4][5] and certain antibiotics components 6,7) as well as in vivo high molecular constituents. [8][9][10] NADase is a membrane-bound ecto-enzyme that is widely distributed in the organism, the richest source generally being the spleen, brain, and liver.…”