THE BIOCHEMICAL FUNCTION OF BIOTIN, VI. CHEMICAL STRUCTURE OF THE CARBOXYLATED ACTIVE SITE OF PROPIONYL CARBOXYLASE

Publisher Website
Google Scholar
Abstract
C14-O2-enzyme was prepared by by incubation of bovine liver mitochondrial propionyl carboxylase for several min. with HC14O3, ATP, and Mg++ and was isolated from the reaction mixture by gel filtration with Sephadex G-50. Carboxyle transfer from C14O2-enzymeto propionyl CoA to form methylmalonyl CoA was stoichiometric with the quantity of biotin present in the C14O2-enzyme (1 mole of carboxyl transferred per mole of biotin). C14O2-enzyme was methylated with CH2N2 and subjected to protoelyticdigestion with Pronase(Streptomyces griseus protease). The fragment released, bearing essentially all of the radioactiveity of the original C14O2-enzyme, wasidentified as N-carbometoxybiocytin. The C14-labelled N-carbomethoxybiocytin was hydrolyzed by the action of pig kidney biotinidase and the C14-labelled hydrolysis product identified as 1[image]-N-carbo-methoxy-(+)-biotin. Therefore, it is concluded that the carboxylated active site of propionyl carboxylase is [epsilon]-N-(1[image]-N-carboxy-(+)-biotinyl)-lysyl-enzyme.