RATE CONSTANTS OF SUGAR-TRANSPORT THROUGH 2 LAMB MUTANTS OF ESCHERICHIA-COLI - COMPARISON WITH WILD-TYPE MALTOPORIN AND LAMB OF SALMONELLA-TYPHIMURIUM

Two LamB (maltoporin) point mutants of Escherichia coli (R8H and Y118F ) and;wild-type LamB of Salmonella typhimurium were reconstituted into artificial lipid bilayer membranes. Ion transport through wild-type L amB of S. typhimurium and the LamB mutants was inhibited by the additi on of carbohydrates of maltose and maltooligosaccharide type in a dose -dependent fashion. The sugar-induced block of the channel function co uld be used for the study of current noise through the different wild- type and mutant LamB-channels. The analysis of the power density spect ra allowed the evaluation of the on and off-reactions (k(1) and k(-1)) of sugar-binding to the binding site inside the channels. Wild-type L amB of S, typhimurium had approximately the same sugar-binding kinetic s as has been observed for LamB of E. coli. The results suggest that t he binding site inside the channel interacts with a maximum of three g lucose residues within the maltooligosaccharides. The LamB mutants R8H and Y118F showed kinetics for sugar binding substantially different f rom that of wild-type LamB. In particular, the on-rate, k(1), for the binding of different sugars of the maltooligosaccharide series to the mutant R8H was approximately 500-times smaller than for wild-type LamB , which resulted in substantially smaller stability constant of sugar binding to the channel. Similarly, the off-rate constant, k(-1), for s ugar binding to the mutant Y118F decreased about 20-fold, which led to a strong increase of the affinity of carbohydrates to the site. The r ole of the amino residues acid R8 and Y118 in the transport of maltose and maltooligosaccharides through LamB-channels is discussed on the b asis of the net flux of sugars through the channels. (C) 1996 Academic Press Limited