THE CATALYTIC MECHANISM OF ADENOSYLHOMOCYSTEINE METHYLTHIOADENOSINE NUCLEOSIDASE FROM ESCHERICHIA-COLI - CHEMICAL EVIDENCE FOR A TRANSITION-STATE WITH A SUBSTANTIAL OXOCARBENIUM CHARACTER/

The substrate and inhibitory specificity of Escherichia coli adenosylh omocysteine (AdoHcy)/methylthioadenosine (MeSAdo) nucleosidase has bee n 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' po sitions in MeSAdo abolished substrate activity. However, the 2'-deoxy analogue of MeSAdo is effective as a substrate; this result provides e vidence against a possible general-base catalysis involving the anchim eric 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 t he enzyme indicate the importance of the glycosidic conformation of th e substrate for binding to the active site. The enzyme discriminates a gainst methanol attack from the solvent during catalysis. This implies the participation of an enzyme-directed water nucleophile. A poor sol vent 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 p H values from 5.0 to 8.5 are similar, with two presumably essential io nisable 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 MeS Ado is substituted by fluorine, a significant decrease (28 500-fold) i n the V-max for enzyme-catalysed hydrolysis of the modi fled substrate is observed. This result indicates a transition state with a substant ial oxocarbenium character. From these data, the reaction mechanism fo r AdoHcy/MeSAdo nucleosidase is discussed.