Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli.
Binding of the substrate analogue p-nitrophenyl alpha-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 introduced by Kennedy et al. [Kennedy, E.P., Rumley, M.V., Armstrong, J.B. (1974) J. Biol. Chem. 249, 33-37]. Two binding sites for NPG were found with dissociation constants of about 16 microM 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.
Lolkema
J S
JS
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Walz
D
D
D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008962:Models, Theoretical; D009004:Monosaccharide Transport Proteins; D009598:Nitrophenylgalactosides / Q000378:metabolism*; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C021499:lactose permease