Alcohol esterification reactions and mechanisms of snake venom 5'-nucleotide phosphodiesterase.
In a previous study we have shown that snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes methanol-esterification reactions [García-Díaz, M., Avalos, M. & Cameselle, J. C. (1991) Eur. J. Biochem. 196, 451-457]. 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-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'-nucleotide esters and agree with the formation of a covalent 5'-nucleotidyl-SVP intermediate susceptible to nucleophilic attack by short-chain (poly)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-O-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 (VH) of substrates yielding AMP and different leaving groups were also assayed. The higher EA and VH values corresponded, respectively, to those acceptors and leaving-group conjugate acids with lower pKa and higher polar-substituent constants (sigma*). The results support the occurrence of general acid-base 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.
García-Díaz
M
M
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Avalos
M
M
Cameselle
J C
JC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000438:Alcohols / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry*; D004952:Esters / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012911:Snakes; D013057:Spectrum Analysis
D000438:Alcohols; D003435:Crotalid Venoms; D004952:Esters; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom