ALCOHOL ESTERIFICATION REACTIONS AND MECHANISMS OF SNAKE-VENOM 5'-NUCLEOTIDE PHOSPHODIESTERASE

In a previous study we have shown that snake venom 5'-nucleotide phosp hodiesterase (SVP) catalyzes methanol-esterification reactions [Garcia -Diaz, M., Avalos, M. & Cameselle, J. C. (1991) Eur J. Biochem. 196, 4 51-457]. Now we have demonstrated that SVP catalyzes AMP transfer from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2-chlo roethanol or 2,2-dichloroethanol. The AMP-O-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'-nucle otide esters and agree with the formation of a covalent 5'-nucleotidyl -SVP intermediate susceptible to nucleophilic attack by short-chain (p oly)alcohols as acceptors alternative to water. To test the kinetic in fluence of the solvent nucleophile in SVP mechanisms. initial rates of ATP solvolysis were assayed in different water/alcohol mixtures. Rela tively high alcohol concentrations inactivated SVP but lower concentra tions gave proportional rates of alcoholysis. An efficiency parameter (E(A)), defined as the ratio of the mole fraction of AMP-O-alkyl ester as a product to that of alcohol as an acceptor in water/alcohol mixtu res, made possible the comparison of alcohols and water as AMP accepto rs at low concentrations, as it could be reasoned that E(A) = 1 for wa ter. Rates of hydrolysis (V(H)) of substrates yielding AMP and differe nt leaving groups were also assayed. The higher E(A) and V(H) values c orresponded, respectively, to those acceptors and leaving-group conjug ate acids with lower pK(a) and higher polar-substituent constants (sig ma). The results support the occurrence of general acid-base catalysi s in the active center of SVP and the identification of rate-limiting steps. A model is proposed for the mechanisms of SVP-catalyzed hydroly sis and alcoholysis which accounts for the influence of the acid-base properties of alcohols on the kinetic profile of SVP reaction sequence s.