Raffinose Metabolism in Escherichia coli K12

Abstract

The utilisation by Edcherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an α-galactosidase specified by the Raf-plasmid D1021. The α-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme, α-Galactosidase hydrolyzes p-nitrophenyl-α-d-galactoside (K_m 0.14 mM), methyl-α-d-galactoside (K_m 30mM), melibiose (K_m, 3.2 mM) and rallinose (K_m 60 mM). The enzymatic activity is strongly inhibited by Ag⁺, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of α-galactosidase with an isoelectric point of 5.1. The purified enzyme has an S_W,2o value of 11.7 ± 0.3S and a molecular weight of 329000 ± 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified α-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of α-galactosidase show enzyme molecules of approximately 11 × 11 nm containing four globular subunits in a tetragonal arrangement. The plasmid-coded α-galactosidase differs from the homologous E. coli enzyme by substrate affinities. cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids