In a previous study we have shown that snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes methanol-esterification reactions [Garcia-Diaz, M., Avalos, M. & Cameselle, J. C. (1991) E m J. Biochem. 196,. Now we have demonstrated that SVP catalyzes AMP transfer from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2-chloroethanol or 2,2-dichloroethanol. The AMP-0-alkyl ester products were identified by HPLC, enzyme analysis, ultraviolet and NMR spectroscopy. Those results show the potential of SVP as a tool to prepare 5'-nucleotide esters and agree with the formation of a covalent 5'-nucleotidyl-SVP intermediate susceptible to nucleophilic attack by short-chain (po1y)alcohols as acceptors alternative to water. To test the kinetic influence of the solvent nucleophile in SVP mechanisms, initial rates of ATP solvolysis were assayed in different water/alcohol mixtures. Relatively high alcohol concentrations inactivated SVP but lower concentrations gave proportional rates of alcoholysis. An efficiency parameter (EA), defined as the ratio of the mole fraction of AMP-0-alkyl ester as a product to that of alcohol as an acceptor in water/alcohol mixtures, made possible the comparison of alcohols and water as AMP acceptors at low concentrations, as it could be reasoned that EA = 1 for water. Rates of hydrolysis (V,) of substrates yielding AMP and different leaving groups were also assayed. The higher EA and V, values corresponded, respectively, to those acceptors and leaving-group conjugate acids with lower pK, and higher polar-substituent constants (a*). The results support the occurrence of general acidbase catalysis in the active center of SVP and the identification of rate-limiting steps. A model is proposed for the mechanisms of SVP-catalyzed hydrolysis and alcoholysis which accounts for the influence of the acid-base properties of alcohols on the kinetic profile of SVP reaction sequences. Enzymes. Phosphodiesterase I (EC 3.1.4.1) ; alkaline phosphatase (EC 3.1.3.1); glycerol kinase (EC 2.7.1.30); lactate dehydrogenase (EC 1.1.1.27); pyruvate kinase (EC 2.7.1.40).enzyme, yielding the leaving group PP, and a covalent intermediate (E-AMP) ; a second nucleophilic displacement by water, giving rise to a non-covalent complex (E 3 AMP); and the dissociation of AMP from the active center. The occurrence of a covalent intermediate and a double-displacement are supported by the isolation of enzyme-bound AMP and TMP [5-71, and by the retention of the stereochemical configuration at phosphorus during the hydrolysis of phosphorothioate analogs or isotopically chiral phosphate diesters L8-121. A threonine residue in the active center has been identified as the site of 5'-nucleotide attachment [13, 141. The participation of the non-covalent complex (E . AMP) is supported by the strong competitive inhibition of the enzyme by AMP and CMP [15].Recently we studied venom and intestine 5'-nucleotide phosphodiesterases in watedmethanol mixtures and found that nucleoside 5'-polyphosphates were converted to a mixture ...

Two rat liver ADP‐ribose pyrophosphatases (ADPRibases) were partially purified. ADPRibase‐I hydrolyzed ADP‐ribose (K m=0.5 μM) giving AMP as a product, required Mg2+ or, less efficiently, Mn2+ (Ca2+ was not active), its activity changed little between pH 7 and 9, and was specific for ADP‐ribose as it did not hydrolyze ADP‐glucose, NAD+, NADH or diadenosine 5′,5″‐P 1,P n ‐n‐phosphates (Ap2A, Ap3A). ADPRibase‐II showed similar properties, except that the K m for ADP‐ribose was 50 μM and may be non‐specific, as the same preparation hydrolyzed ADP‐glucose, NADH and Ap2A. ADPRibase‐I fulfils the requirements of a specific turnover pathway consistent with a cellular role for free ADP‐ribose.

SummaryAlthough systemic vasculitis can be a complication of inflammatory bowel disease at several locations (skin, eyes, brain, mesentery, and lung) the association of retinal vasculitis with Crohn's disease is rare. We studied a 26-year-old woman with biopsy-demonstrated Crohn's disease who developed a severe bilateral retinal arteritis and phlebitis, with acute loss of vision.

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...

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...