The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli

Abstract

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...

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

“…An intriguing question addressed by the protonation states of the adenine product is the identity of the specific chemical group that donates a proton to N9. It has been proposed that the conserved residue E13 acts as a general base to activate the nucleophilic water molecule that attacks the intermediate during the enzymatic reaction (9,12,33). Examination of the structure of the ternary product complex shows that the protonated N9 is 6.7 Å from E13 and lacks neighboring proton acceptors that could allow a proton shuttle-like mechanism.…”

“…The new orientation of the deuterium atom for the O3′ hydroxyl allows a new O-D···O hydrogen-bond interaction to form with the O2′ hydroxyl at a distance of 2.1 Å. The O-D···Oδ2 hydrogenbond interaction between the O3′ hydroxyl and the carboxylate side chain of E175 was shown to be essential for the catalytic reaction, because the removal of the O3′ hydroxyl from the ribose moiety and the use of E175 EcMTAN variants eliminate catalytic activity (6,9). Based on these results, it has been proposed that the O3′ hydroxyl becomes ionized during the formation of the transition state to assist in stabilizing the oxocarbenium ion intermediate (11,32), but the neutron structures presented here demonstrate a protonated O3′ hydroxyl and a fully deprotonated E175 carboxylate.…”

“…1B) (8)(9)(10)(11)(12)(13). Catalysis is initiated by protonation of N7 of the adenine moiety by an aspartic acid residue, D198.…”

“…Maintaining the protonated form of D198 requires elevation of the side chain pK a to a level much higher than the theoretical pK a of an aspartic acid side chain. Indeed, assessment of side-chain ionization of the analogous residue in the Escherichia coli homolog determined a pK a of 8.2, which can be attributed to the burial of the D198 side chain upon substrate binding (9,14). Additionally, D198 has been demonstrated to be essential for the enzymatic activity through the use of an asparagine variant (D198N) of HpMTAN that binds substrate but does not promote hydrolysis (5,12,15).…”

“…In previous studies of E. coli MTAN (EcMTAN), conserved residues E12 and E175 were shown to be essential for the catalytic reaction based on inactive variants (6). Furthermore, it was suggested that E12 functions as a general base by activating the nucleophilic water molecule, because a second ionizable group with a pK a value of 5.6 was identified (9).…”