Enzymatic Conversion of Vitamin B12s to a Cobamide Coenzyme, α-(5,6-Dimethylbenzimidazolyl)deoxyadenosylcobamide (Adenosyl-B12)

Abstract

Abstract 1. Clostridium tetanomorphum contains an adenosylating enzyme, that converts B12s (a reduced form of vitamin B12 in which the cobalt atom is in the +1 oxidation state) to the cobamide coenzyme, adenosyl-B12 (B12s + adenosine triphosphate → adenosyl-B12 + tripolyphosphate). 2. The enzyme has been purified approximately 300-fold, with an over-all recovery of about 40% by treatment of sonically disrupted cells with protamine, followed by selective heat denaturation and chromatography on diethylaminoethyl cellulose. The purified enzyme is free from B12a reductase and glutamate mutase. 3. The pH optimum for the enzyme is 8.0. Michaelis constants for B12s and ATP are 1.0 and 1.6 x 10-5 m, respectively. B12s cannot be replaced by B12r or B12a (forms of B12 in which the oxidation state of cobalt is +2 and +3, respectively). ATP can be replaced by cytidine triphosphate and, to a lesser degree, by the triphosphates of uridine, guanosine, and inosine. Mn++ is required as divalent cation; Co++ and Mg++ are less effective. 4. With the purified enzyme the forward reaction can be demonstrated spectrophotometrically by means of the increase in absorbance at 525 mµ as B12s is converted to adenosyl-B12. No evidence could be obtained for reversal of the reaction.