Oligonucleotides composed of a phosphorylated d-altritol backbone with nucleobases introduced in the 2'-position, hybridise strongly and sequence-selectively with RNA in an antiparallel way. The order of hybridisation strength is: dsANA b ANA:RNA b ANA:DNA. Complexes between ANA and RNA or DNA are more stable than between HNA and natural oligonucleotides. The dsANA hybrid is extremely stable. When compared with an identical dsDNA sequence, the DT m per modification is 10.2 8C for a hexamer. CD spectral analysis indicates that ANA complexes are very similar to the A-form dsRNA duplex. ANA are stable in alkaline medium up to pH 12 and, likewise, are not degraded in human serum.

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′-A-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 V max . Plots of log V max and log (V max /K m ) 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 pK a values of 5.6 and 8.2, whereas K m is independent of pH. When the 2′-A-hydroxy group of MeSAdo is substituted by fluorine, a significant decrease (28 500-fold) in the V max 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.Keywords : adenosylhomocysteine nucleosidase ; substrate; inhibitor ; catalytic mechanism.S-Adenosylhomocysteine (AdoHcy) is the product of biological transmethylation reactions that utilise S-adenosylmethionine (AdoMet) as a methyl donor [1,2]. In various microorganisms (bacteria and protozoa), this compound and 5′-methylthioadenosine (MeSAdo), formed from adenosylmethionine by several independent pathways [3], are substrates of AdoHcy/ MeSAdo nucleosidase. This enzyme catalyses the cleavage of the metabolites into S-ribosylhomocysteine and methylthioribose, respectively, adenine also being generated by both reactions [4,5].AdoHcy/MeSAdo nucleosidase is involved in a variety of biological processes. In particular it is required for the regeneration of free Hcy from AdoHcy in various prokaryotes [5,6], and plays a significant role in the metabolism of these microorganisms via the regulation of the MeSAdo concentration (a potent inhibitor of spermidine and spermine synthases) [7Ϫ9]. In microorganisms in which MeSAdo phosphorylase is missing, AdoHcy/MeSAdo nucleosidase is essential for methionine salvage [10Ϫ12].Correspondence to G. Guillerm, Laboratoire de Chimie Bioorganique, U.M.R. 6519, U.F.R. Sciences de Reims, Moulin de la Housse, B.P. 1039, F-51687 Reims cedex 2, France Fax: ϩ33 3 26 05 31 66. E-mail: georges.guillerm@univ-reims.fr Abbreviations. AdoHcy, S-adenosylhomocysteine; MeSAdo, 5′-d...

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