It is not known whether the enzymes 5'-nucleotide phosphodiesterase/nucleotide pyrophosphatase (EC 3.1.4.1/ EC 3.6.1.9) catalyze the transfer of nucleotides to acceptors other than water. We have investigated the action of snake venom and bovine intestinal mucosa phosphodiesterases on nucleoside 5'-polyphosphates in the presence of methanol. In those conditions, GTP was converted by snake venom phosphodiesterase to a mixture of GMP and another compound with a different retention time in reverse-phase high-performance liquid chromatography. That compound, by ultraviolet, 'H-and I3C-nuclear magnetic resonance spectroscopic analysis, and by enzyme analysis, was characterized as the methyl ester of GMP (GMP-OMe). The molar fraction [GMP-OMe]/ [GMP + GMP-OMe] formed was higher than the molar fraction of methanol as a solvent in reaction mixtures.Similar reactions took place at comparable rates with snake venom and bovine intestinal mucosa phosphodiesterases using several nucleoside 5'-polyphosphates as substrates. The ability of 5'-nucleotide phosphodiesterases to catalyze transfer reactions to a non-water acceptor is relevant to the mechanism of the enzymes, to their use as analytical tools, and to their possible use/role in the preparativelin vivo synthesis of nucleotide esters.Phosphodiesterase I or 5'-nucleotide phosphodiesterase, and nucleotide pyrophosphatase are overlapping names which, in many cases, can be applied to the same enzyme. For example, snake venom phosphodiesterase (SVP) [l], 5'-nucleotide phosphodiesterase from bovine intestinal mucosa (BIMP) [2, 31, and liver nucleotide pyrophosphatase [4, 51 show both types of activity: they are phosphohydrolases removing 5'-nucleotides from phosphodiester and phosphoanhydride linkages. The mechanism of action of SVP [6 -91 and BIMP [lo-141 involves the formation of a nucleotidyl intermediate, covalently bound to the enzyme, which is transferred to a molecule of water, yielding the 5'-nucleotide hydrolysis product.The possibility that 5'-nucleotide phosphodiesterase/ nucleotide pyrophosphatase enzymes catalyze 5'-nucleotidyl transfers to acceptors other than water has been little explored. On the one hand, there are quotations in the literature indicating that transfer reactions of BIMP have been searched for but not detected [13, 151. In addition, the participation of rat liver nucleotide pyrophosphatase in the transfer of 5'-adenylyl groups to glycerol had been deemed possible, but it was disregarded on kinetic grounds [16]. On the other hand, there is an early report studying the action of Habu snake venom and pig kidney phosphodiesterase preparations on the artificial substrate ethyl-phospho-p-nitrophenol [17]. In that case, incubations in the presence of glycerol or ethylenglycol yielded a lower molar amount of ethyl phosphate (measured as phosphatase-labile phosphate) than of p-nitrophenol, an indication that transfers to alcohol acceptors could be taking place, although the esterification products were not directly detected [17]. Altogether, the scarce a...