Abstract
Five analogs of adenosylcobalamin modified in the adenine moiety of the Co beta ligand were synthesized and tested for coenzymic function with diol dehydrase of Klebsiella pneumoniae ATCC 8724. 1-Deaza and 3-deaza analogs of adenosylcobalamin were active as coenzyme, whereas 7-deaza and N6,N6-dimethyl derivatives and guanosylcobalamin did not show detectable coenzymic activity. 7-Deaza and N6,N6-dimethyl analogs acted as strong competitive inhibitors with respect to adenosylcobalamin. The formation of cob(II)alamin as intermediate in the catalytic reaction was spectroscopically observed with catalytically active complexes of the enzyme with 1-deaza and 3-deaza analogs in the presence of 1,2-propanediol, but not with complexes with the inactive analogs. Oxygen sensitivity of the enzyme-analog complexes suggests that the carbon-cobalt bond of 1-deaza and 3-deaza analogs becomes activated by the enzyme even in the absence of substrate. These results indicate that the importance of the nitrogen atoms in the adenine moiety of the coenzyme for manifestation of catalytic function and for activation of the carbon-cobalt bond decreases in the following order: N-7 greater than 6-NH2 greater than N-3 greater than N-1. The dissociation constant for 5'-deoxyadenosine determined by equilibrium dialysis at 37 degrees C was about 23 microM.
Highlights
The Synthesisof Adenine-modified Analogs of Adenosylcobalamin and Their Coenzymic Function in the Reaction Catalyzed by Diol Dehydrase*
Five analogs of adenosylcobalamin modified in the During the course of our studies addressed to answer this adenine moietyof the Cos liganwdere synthesized and question, we reached the conclusion that the interactions of tested for coenzymic function with diol dehydrase of diol dehydrase with peripheral amide side chains of the coen
Klebsiella pneumoniae ATCC 8724. 1-Deaza and 3- zyme [5, 6], which could lead to distortion of the corrin ring, deazaanalogsofadenosylcobalaminwere active as as well as with the adenosyl moiety [4, 7,8,9] are essential for coenzyme, whereas 7-deaza and N‘,W-dimethyl de- the activation of the carbon-cobalt bond
Summary
Relative mobility in paper electrophoresis a t pH 2.7b unit is defined asthe amount of enzyme activity catalyzing the formation of 1 pmol of propionaldehyde/min under the standard conditions. Mobility of CN-Cbl and aquacobalamin was taken as 0 and 1, determined spectrophotometrically after converting them to dicyan- respectively. Synthesis of 5'-Chloro-5'-deoxyribonucleosides-5'-Chloro-5'-deoxyribonucleosides were synthesized from the corresponding nucleosides by reaction with SOClp in hexamethylphosphoramide,as de-. &.Cl.A& for 5'-chloro-5'-deoxy-N6,N6-dimethyladenosine in solvent system E was 1.12. All five analogs were synthesized from the corresponding 5'-chloro-5'-deoxyribonucleosidesby an established procedure [16, 30]. Each 5'-chloro-5'-deoxyribonucleoside was reacted with cob[1]alamin whichwas formed from CN-Cbl by reduction with NaBHl orzinc powder/NH&l. The coenzyme analogs formed were purified by phosphocellulose (H+form) column chromatography and decomposition, catalytic reduction (Hp/PtOz),etc., followed by TLC preparative thin-layer chromatography on silica gel using solvent analyses of the nucleosides or organic bases formed.
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