Abstract
The Enzymatic Synthesis of Cobamide Coenzymes
Highlights
Purijcation of Cobamide Coenzyme-synthesizing Enzyme-The soluble supernatant solution obtained by sonic oscillation of washed cells of P. shermanii was fractionated by precipitation between 32 and 40 To saturation with solid ammonium sulfate at pH 6.5
The biosynthesis of cobamide coenzymes was investigated in an enzyme preparation obtained from Propionibacterium shermanii
The formation of cobamide coenzymes requires the presence of adenosine triphosphate (ATP), Ill&+, K+, reduced flavin adenine dinucleotide, a sulfhydryl compound, and a cobamide derivative
Summary
The conditions of growth of P. shermanii and the preparation of cell-free extracts have been described in the preceding communication [7]. The reaction mixture used to assay for the coenzyme-synthesizing activity contained 5 pmoles of potassium phosphate buffer, pH 7.0, 150 mpmoles of ATP (sodium salt), 150 mpmoles of MnC12, 25 mpmoles of DPNH, 2 mpmoles of hydroso-BC,l 2.5 mpmoles of F,4D, 0.3 pmole of 2-mercaptoethanol, 0.5 pmole of GSH, and diaphorase (3 pg of protein) in a final volume of 50 ~1. The mixture was centrifuged, and the supernatant solution was neutralized with 8 ~1 of 14% KOH. The amount of BC coenzyme was determined in a 50.~1 aliquot of the supernatant solution by the spectrophotometric glutamate isomerase assay [12]. One unit of coenzymesynthesizing activity is defined as the amount of enzyme required to catalyze the synthesis of 1 mpmole of BC coenzyme in 1 hour under the above assay conditions
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