Release of inorganic tripolyphosphate from adenosine triphosphate during vitamin B-12 coenzyme biosynthesis.

The enzymatic conversion of vitamin Ba to its coenzyme form has been described in cell-free systems derived from Propionibacterium shermanii (1) and from Clostridium tetanomorphum (2). The 5’-deoxyadenosyl moiety that replaces the cobalt-bound cyanide of cyanocobalamin during Blz coenzyme formation is derived from adenosine triphosphate (ATP) (3). In the present work, the fate of the phosphate residues of ATP during Blz coenzyme synthesis has been examined. It is demonstrated that all three phosphate groups of ATP are released as inorganic tripolyphosphate. Synthesis of coenzyme from the vitamin requires, in addition to ATP, a sulfhydryl compound and a reduced flavin (1). Our previous studies (4) have established that cyanide is released from vitamin Blz only in the presence of the complete system for coenzyme synthesis. In addition, during the process of coenzyme synthesis, there appears to be no intermediate reduction of vitamin Blz to BIZ,,~ a derivative of the vitamin in which the valence of cobalt has been reduced from +3 to +2. The present study indicates that vitamin B12,, although not an obligate intermediate in the reaction, can serve as a substrate for coenzyme synthesis. It is noteworthy that, even when vitamin B1zr is used as a substrate, there is still a requirement for reducing agent. These data suggest that the enzyme that catalyzes the synthesis of Blz coenzyme requires a transfer of 2 electrons (Equation 1).