| Abstract: | Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme. |
