High Efficiency of Glycerol 2‐Phosphate and sn ‐Glycerol 3‐Phosphate as Nucleotidyl Acceptors in Snake Venom Phosphodiesterase Esterifications

Abstract

Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP by primary R-CH,OH alcohols with uncharged R residues, yielding AMP-0-CH,R esterification products. The alcohols compete with water for an SVP-bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2-phosphate and sn-glycerol 3-phosphate with ATP to yield AMP-Oglycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP-0-glyceryl esters by alkaline phosphatase. The results demonstrated that R-CH,OH alcohols with negatively charged R residues, as well as secondary alcohols, act as adenylyl acceptors in SVP reactions, thus extending the usefulness of SVP as a tool to produce 5'-nucleotide derivatives. The efficiencies (E,) of glycerol phosphates as adenylyl acceptors were very high at low, millimolar concentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2-phosphate, when P, or NaCl was present. In contrast, glycerol E, was independent of its own concentration, P,, and NaCI. The responses of glycerol phosphates indicate that they act as adenylyl acceptors via a mechanism different from uncharged R-CH,OH alcohols. The occurrence of an acceptor-binding enzyme site, specific for negatively charged R residues, and its potential relevance to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucleotidyl transferases are discussed.Keyvvords: phosphodiesterase-I ; nucleotide-pyrophosphatase ; esterification ; 5'-nucleotide esters ; glycerol phosphates.Snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes the solvolysis of phosphodiester and phosphoanhydride derivatives of 5'-nucleotides. The reaction sequence includes a double displacement, with the formation of a 5'-nucleotidyl intermediate covalently bound to the enzyme 11-51 and its transfer to an acceptor that can be water or an R-CH,OH alcohol [6, 71. The solvolysis of ATP in water/alcohol mixtures follows a branched route (Scheme 1) 171.Reaction rates with different acceptors may reflect both the reactivities of the alcohols and their influence on enzyme structure and activity. Both of these properties depend on the nature and concentration of the alcohol but are unrelated to each other. Therefore, to compare acceptor reactivities without interference by enzyme (in)activation effects, the parameter EA (alcohol acceptor efficiency) is very convenient (71 :For an alcohol as reactive as water, E, = 1. since in this case the mole fraction of the esterification product AMP-0-CH,R equals the mole fraction of alcohol as an acceptor.Comparisons of E, values disclose valuable mechanistic information. Short-chain R-CH,OH alcohols with uncharged R residues, display E,% values that are largely concentration independent, but depend on the nature of R. For alcohols with a hydroxyl group pK., 3 15.5, E, relates to electronic factors like the acid strength: t...

Use of potato tuber nucleotide pyrophosphatase to synthesize adenosine 5′-monophosphate methyl ester: Evidence that the solvolytic preferences of the enzyme are regulated by pH and temperature

“…For those reasons, AppA was used as the substrate, instead of ATP, employed in studies of SVP-catalyzed alcoholysis (García-Díaz et al, 1993;Vergeles et al, 1995).…”

“…SVP reactions in alcohol-water mixtures have been studied only at pH 7.5 and 37°C (García-Díaz et al, 1993;Vergeles et al, 1995). To elucidate whether pH and temperature effects on PNP could be a general feature of nucleotide pyrophosphatases, their effects on SVP were also studied.…”

“…This enzyme is commonly used as an analytical tool for the struc-tural characterization of nucleotidic compounds. More recently, SVP has also been used for the preparation of small amounts of alkyl esters of 5Ј-nucleotides, as the enzyme admits alcohols as solvolytic agents (García-Díaz et al, 1993;Vergeles et al, 1995). We are interested in the synthesis of these esters because they are pharmacologically important as potential (ant)agonists of purinoceptors or other receptors responding to nucleotides (Abbracio and Burnstock, 1994;Fredholm et al, 1994), and inhibitors of enzymes involved in the turnover of nucleotides (Ziganshin et al, 1994;Zimmermann, 1992).…”

Rat liver nucleotide pyrophosphatase/phosphodiesterase is an efficient adenylyl transferase

“…It was obtained in a partially purified form, which generated several protein bands when analysed by SDS\PAGE or by native gel electrophoresis stained with Coomassie Blue, but only one band was produced when native gels were stained for NPP\PDE activity with either 2-naphthyl phenylphosphonate [28] or 1naphthyl-5h-dTMP [29] (Sigma, Madrid, Spain) as the substrate. Other materials were from sources reported previously [25,26].…”

“…the basis of the knowledge that snake venom and mammalian NPP\PDE enzymes catalyse a double nucleophilic displacement on the α-phosphorus atom of the adenylyl moiety of ATP : a threonine lateral chain carries out first an attack that leads to an adenylyl-enzyme intermediate ; thereafter, a molecule of water splits the phosphodiester linkage of this intermediate, releasing AMP as a product of hydrolysis [16,[19][20][21][22][23]. In this mechanism, one may ask whether, in the active centre of mammalian NPP\PDE, alcohols could substitute for water, as is known to occur in the active centre of snake venom NPP\PDE [24][25][26]. From the present study, rat NPP\PDE has been identified as a more efficient adenylyl transferase than the snake enzyme, with an active centre capable of establishing multiple interactions with specific alcohol acceptors of the enzyme-bound adenylate.…”