A soluble enzyme system has been isolated from biotin-deficient rat liver acetone powder which catalyzes the synthesis of propionyl holocarboxylase from d-biotin and its apocarboxylase, in the presence of ATP and Mg++ ions. The enzyme system has been partially purified by (NH₄)₂SO₄ precipitation and resolved into two obligatory components by alumina C y gel fractionation. The gel supernatant fraction has been further purified by DEAE cellulose ion-exchange chromatography. Since the active component in the gel supernatant fraction and the endogenous propionyl holocarboxylase have the same elution pattern when chromatographed on DEAE cellulose, it appears likely that the gel supernatant contains the propionyl apocarboxylase. The gel eluate most likely contains an enzyme which catalyzes the covalent bonding or d-biotin to propionyl apocarboxylase and other proteins.

In order to measure the activity of the propionyl holocarboxylase synthesizing system, two assay procedures were used. One assay procedure employed the incorporation of biotin-1-c¹⁴ into protein as a measure or the activity of the enzyme system. The other assay was based upon the biotin and ATP dependent increase of propionyl carboxylase activity catalyzed by the enzyme system. Both procedures gave similar results.

Propionyl holocarboxylase formation was found to be ATP specific since neither CTP, OTP, ITP, nor UTP could replace ATP. In addition, a mixture or all five nucleoside triphosphates was no more effective than ATP alone. Versene inhibited the reaction and MgCl₂ was able to reverse this inhibition, indicating a Mg++ ion requirement.

The ability of various biotin derivatives to replace d-biotin in propionyl holocarboxylase formation was investigated. It was round that if either the valeric acid side chain is altered. as in homo- or nor-biotin, of if the sulfur atom is removed or substituted, as in desthiobiotin or oxybiotin, holocarboxylase formation did not occur. Similarly, neither of these derivatives inhibited the bonding of c¹⁴-biotin to protein. Biocytin has been eliminated as an intermediate in propionyl holocarboxylase formation.

Hydroxylamine does not inhibit the reaction. nor is CoA required. These data would appear to eliminate the involvement of free carboxyl activated biotinyl intermediates in the formation of the holocarboxylase from its apocarboxylase and biotin. Although the evidence suggests a concerted mechanism for the reaction, mechanisms involving enzyme bound intermediates are not completely eliminated by these data.

In order to determine the nature of the attachment of biotin to propionyl carboxylase, c¹⁴-biotin labeled propionyl carboxylase was prepared. The labeled carboxylase was enzymatically hydrolyzed and chromatographed on Whatman 3MM paper. The biocytin peak contained nearly 100% of the radioactivity recovered. This peak was eluted and rechromatographed by ion-exchange chromatography. The only radioactive component obtained by this procedure was biocytin.

The data presented indicate that the propionyl holocarboxylase synthesizing system catalyzes the ATP dependent covalent bonding of d-biotin to the lysyl-(-amino groups or propionyl apocarboxylase.