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...
Rat liver nucleotide pyrophosphatase/phosphodiesterase I (NPP/PDE) catalysed efficiently the transfer of adenylate from ATP to alcohols (methanol, ethanol, propanol, ethylene glycol, glycerol, 2, 2-dichloroethanol and glycerol 2-phosphate), which acted as adenylate acceptors competing with water with different efficiencies. NPP/PDE kinetics in alcohol/water mixtures were accounted for by rate equations for competitive substrates, modified to include alcohol negative co-operativity and, depending on the nature of the alcohol, enzyme denaturation by high alcohol concentrations or activation by low alcohol concentrations. The correlation of alcohol efficiencies with alcohol acidities, the comparison of rat liver with snake venom NPP/PDE, and the different effects of ionic additives on the efficiencies of glycerol 2-phosphate and glycerol provided evidence for interaction of the alcohols with a base catalyst, a non-polar and a cationic subsite in the active centre of rat liver NPP/PDE. The enzyme thus appears to be well suited to act as transferase, and we propose that NPP/PDE could be an adenylylating agent in the membrane.
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