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The substrate and inhibitory specificity of Escherichia coli adenosylhomocysteine (AdoHcy)/methylthioadenosine (MeSAdo) nucleosidase has been explored with several MeSAdo analogues modified on the sugar moiety at the 2', 3' and 5' positions. Alteration at C3' or at C2' and C3' positions in MeSAdo abolished substrate activity. However, the 2'-deoxy analogue of MeSAdo is effective as a substrate; this result provides evidence against a possible general-base catalysis involving the anchimeric assistance of the 2'-alpha-hydroxy group and the formation of a 1,2-epoxide as an intermediate in the catalytic process. The results of a study of the interaction of an 8,5'-cyclo analogue of MeSAdo with the enzyme indicate the importance of the glycosidic conformation of the substrate for binding to the active site. The enzyme discriminates against methanol attack from the solvent during catalysis. This implies the participation of an enzyme-directed water nucleophile. A poor solvent kinetic deuterium-isotope effect was measured (0.93) on the Vmax. Plots of log Vmax and log (Vmax/Km) for MeSAdo as a function of pH values from 5.0 to 8.5 are similar, with two presumably essential ionisable groups for catalysis with apparent pKa values of 5.6 and 8.2, whereas Km is independent of pH. When the 2'-alpha-hydroxy group of MeSAdo is substituted by fluorine, a significant decrease (28 500-fold) in the Vmax for enzyme-catalysed hydrolysis of the modified substrate is observed. This result indicates a transition state with a substantial oxocarbenium character. From these data, the reaction mechanism for AdoHcy/MeSAdo nucleosidase is discussed.
Allart et al. (1998) studied this question.