Thiamine transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide.
Active transport of thiamin (vitamin B(1)) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K(M)=15 nM and V(max)=46 U mg(-1). Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V(max) by approximately 50% without effecting K(M) or the apparent first-order rate constant (k(obsd)). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.
Hollenbach
Andrew D
AD
Department of Biochemistry and Molecular Biology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205, USA. andrew.hollenbach@stjude.org
Dickson
Kimberly A
KA
Washabaugh
Michael W
MW
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D013831:Thiamine / Q000032:analysis / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000255:Adenosine Triphosphate; D003545:Cysteine; D005033:Ethylmaleimide; D013831:Thiamine