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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′-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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A direct method for the preparation of 5'-S-alkynyl-5'-thioadenosine and 5'-S-allenyl-5'-thioadenosine has been developed. Treatment of a protected 5'-acetylthio-5'-deoxyadenosine with sodium methoxide and propargyl bromide followed by deprotection gave the 5'-S-propargyl-5'-thioadenosine 4. Under controlled base-catalysis with sodium tert-butoxide in tert-butyl alcohol 4 was quantitatively converted into 5'-S-allenyl-5'-thioadenosine 5 or 5'-S-propynyl-5'-thioadenosine 6. Incubation of recombinant human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 microM, respectively). Compound 4 caused complete conversion of the enzyme from its E-NAD(+) to E-NADH form during the inactivation process. This indicates that 4 is a substrate for the 3'-oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, the NAD(+)/NADH content of the enzyme was not affected during the inactivation process with 5 and 6, and their mechanism of inactivation was further investigated. Addition of enzyme-sequestered water on the S-allenylthio group of 5 or S-propynylthio group of 6 within the active site should lead to the formation of the corresponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated protein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MS(n) experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivated AdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activity of 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC(50): 1.9 microM) was noted with 5 against vaccinia virus.