Effect of cyanide on the biosynthesis of ascorbic acid by an enzyme preparation from goat-liver tissue

Abstract

Potassium cyanide (50 mM), without any added cofactor, greatly stimulates the biosynthesis of L-ascorbic acid from D-glucuronolactone by an enzyme preparation from goat-liver extract containing the microsomes and the soluble supernatant. The enzyme preparation can also convert L-gulonolactone into L-ascorbic acid with or without the presence of cyanide and in the absence of any added co-factor. But conversion is greater in the absence of cyanide. The dialyz-ed enzyme concentrate retains its full activity. Addition of cofactors such as di- or tri-phosphopyridine nucleotide, reduced di- or tri-phosphopyridine nucleotide, nicotinamide, magnesium chloride, coenzyme A, glucose 1-phosphate, glucose 6-phosphate and flavinadenine di- and mono-nucleotide does not further enhance the synthesis. In the presence of cyanide only the lactone forms of the substrates are active. Neither the sodium salts of the free acids nor any other substrate tried are converted into ascorbic acid under the conditions studied. The conversion of D-glucuronolactone is inhibited by p-chloro-mercuribenziate a.nd this inhibition is reversed by reduced glutathione, indicating the probability of the involvement of some thiol groups in the enzyme system concerned in the cyanide-mediated synthesis. Adenosine triphosphate and uridine triphosphate are strikingly inhibitory to the synthesis of ascorbic acid from D-glucuronolactone and L-gulonolactone in vitro in the presence of cyanide. The conversion of D-glucuronolactone into L-ascorbic acid in the presence of cyanide is not inhibited by sodium fluoride, sodium azide, 2,4-dinitrophenol, phlorizin, arsenite, arsenate or pyrophosphate. Cyanide cannot be replaced by sodium azide, carbon monoxide and reducing substances such as reduced glutathione, sodium dithionite or sodium hypophosphite for the conversion of D-glucuronolactone into L-ascorbic acid. The presence of borate or Antabuse, each at mM-concentration, inhibits the conversion of D-glucuronolactone into L-ascorbic acid to about 50%. The optimum conditions for the action of the enzyme system were studied. The biosynthesized ascorbic acid was identified by paper chromatography and by a biological test with one guinea pig.