Binding of p-nitrophenyl-.alpha.-D-galactopyranoside to lac permease of Escherichia coli

Abstract
Binding of the substrate analogue p-nitrophenyl α-D-galactopyranoside (NPG) to lac permease of Escherichia coli in different membrane preparations was investigated. Binding was assayed with an improved version of the centrifugation technique earlier introduced. Two binding sites for NPG were found with dissociation constants of about 16 µM and 1.6 mM at pH 7.5 and room temperature. With purified lac permease reconstituted into proteoliposomes, it could be shown that one permease molecule binds two substrate molecules. Oxidation of lac permease with the lipophilic quinone plumbagin or alkylation with the sulfhydryl reagent N-ethylmaleimide caused a 12-fold increase in the first dissociation constant. The second dissociation constant seemed to be increased as well, but its value could not reliably be estimated. Ethoxyformylation of lac permease with diethyl pyrocarbonate totally abolished NPG binding. The implications of these results for the catalytic performance of the enzyme are discussed

This publication has 0 references indexed in Scilit: