Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma-glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: Insights into the enhanced Co-S bond stability of the natural product glutathionylcobalamin

Glutathionylcobalamin (gamma -glutamylcysteinylglycinylcobalamin; gamma -Gl uCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B-12 coenzymes adenosylcobalamin and methylcoba lamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the greater t han or equal to6 x 10(4) fold less stable cysteinylcobalamin, Cys-Cbl. In o rder to determine which parts of the glutathione tripeptide contribute to t he overall stability of glutathionylcobalamin, two cysteine-containing dipe ptides, which are truncated versions of glutathione, were used to synthesiz e their corresponding cobalamins, specifically gamma -glutamylcysteinylCb1 (gamma -GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with gl utathionylCbl, the dipeptide gamma -GluCys-Cbl forms a stable thiolatocobal amin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutath ionylcobalamin and its congeners, compared to cysteinylcobalamin and its an alogues, must be derived from destabilization by the gamma -NH3+ group in c ysteinylcobalamin, or stabilization by the gamma -NHC(=O)- amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in gamma -GluCys-containing cobalamins, gam ma -GluCys-Cbl was crystallized and yielded the first X-ray structural dete rmination of a true thiolalocobalamin, and only the second structure of a c obalamin containing a Co-S bond, the first example being Randaccio and co-w orkers' 1999 structure of the thioketone complex, thioureacobalamin, (NH2)( 2)CSCbl. Key features of the structure of gamma -glutamylcysteinylcobalanli n include (i) a normal Co-S bond length of 2.267(2) Angstrom, (ii) a Co-N(a xial) bond length of 2.049(6) Angstrom, (iii) two alternate conformations o f the gamma -glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2 degrees, the highest degree of folding yet observed for a co balamin. These results do not show any strong stabilization (e.g., no short ened Co-S bond), although it is not clear for certain what the effect is (s tabilizing or destabilizing) of the elongated Co-N(axial) bond; instead, th e crystallographic results suggest that the metastable Cys-Cbl probably has a Co-S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co-S bond). Overall, the results s trongly suggest that placing a positive charge on the gamma -NH3+ stabilize s the Co-S bond cleavage transition state, thereby setting the stage for th e needed full thermolysis product and kinetic studies-as a function of the axial-base on-off equilibrium-that will be required to understand in even g reater detail the unique stability of glutathionyl- (gamma -glutamylcystein ylglycinyl-) and gamma -glutamylcysteinylcobalamins.