Acetyl Coenzyme A Carboxylase System of Escherichia coli

Abstract

Abstract Methylation of enzymatically synthesized carboxy-d-biotin yields 1'-N-methoxycarbonyl-d-biotin methyl ester, a fact which earlier led to the conclusion that the site of carboxylation of biotin was the 1'-N position. However, as pointed out by Bruice and Hegarthy ((1970) Proc. Nat. Acad. Sci. U.S.A. 65, 805) this structural assignment is equivocal for if carboxylation were to occur at the ureido-O, methylation would facilitate intramolecular O to N migration of the (methoxycarbonyl) group and lead to the same derivative, 1'-N-methoxycarbonyl-d-biotin methyl ester. In the present investigation definitive evidence was obtained which indicates that the ureido-N of biotin is in fact the site of carboxylation. Carboxy-d-biotins generated enzymatically by the biotin carboxylase or carboxyltransferase components of the Escherichia coli acetyl-CoA carboxylase system and tested immediately have stability properties indistinguishable from that of 1'-N-carboxy-d-biotin; first-order decarboxylation rates over the pH range 5 to 9 are identical for the enzymatic products and the authentic carboxybiotin derivative. Specificity studies reveal that the carboxylate terminus of the valeric acid side chain of biotin renders this molecule a poor model substrate for the E. coli carboxyltransferase; it accounts as well for the inability of this enzyme to utilize 1'-N-carboxy-d-biotin as a carboxyl donor substrate. Thus, derivatives lacking the side chain carboxylate function, e.g. biotinol and biotinyl acetate, which are excellent carboxyl acceptor substrates, were used as precursors for the synthesis of enzymatically active 1'-N carboxy derivatives. The carboxyltransferase-catalyzed reaction of [3-14C]malonyl-CoA and d-biotinyl acetate yields a product which after methylation and chromatography has properties identical with 1'-N-methoxycarbonyl-d-biotinyl acetate. Alkaline hydrolysis of the labeled compound yielded putative 1'-N-[14C]carboxy-dbiotinol which serves as a substrate for E. coli carboxyltransferase. 1'-N-Methoxycarbonyl-d-biotinyl acetate was chemically synthesized from d-biotinyl acetate and methylchloroformate under conditions known to effect 1'-N-acylation of a related d-biotin derivative. The product was characterized by chemical, mass spectral, and nuclear magnetic resonance (proton and 13C NMR) analyses; 13C NMR spectra indicated methoxycarbonyl substitution at the 1'-N, rather than the ureido-O, of the imidazolidone ring. Alkaline hydrolysis of 1'-N-methoxycarbonyl-d-biotinyl acetate yields a product, 1'-N-carboxy-d-biotinol, which served efficiently as a carboxyl donor substrate for carboxyltransferase-catalyzed transcarboxylation to acetyl-CoA giving rise to malonyl-CoA. These results and the fact that authentic 1'-N-carboxy-d-biotin also is a substrate for biotin carboxylase-catalyzed net ATP formation from ADP and Pi indicate that the 1'-ureido-N position of biotin is the enzymatic site of carboxylation.